{"gene":"SLIT2","run_date":"2026-06-10T07:46:35","timeline":{"discoveries":[{"year":1999,"finding":"Human Slit2 acts as a chemorepellent for olfactory and hippocampal axons and collapses their growth cones, functioning through Robo1 and Robo2 receptors in the developing forebrain.","method":"Collagen gel repulsion assay, growth cone collapse assay using recombinant human Slit2","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro functional assay with recombinant protein, replicated across multiple axon types, consistent with Drosophila genetic data","pmids":["10197527"],"is_preprint":false},{"year":2001,"finding":"Slit2 is proteolytically cleaved in vivo into a 140 kDa N-terminal fragment (Slit2-N) and a 55-60 kDa C-terminal fragment (Slit2-C). Only Slit2-N (and uncleaved Slit2-U) bind Robo receptors. Slit2-N repels olfactory bulb axons and induces growth cone collapse, while only Slit2-N (not Slit2-C) induces DRG axon branching. The full-length uncleaved form antagonizes branching by Slit2-N.","method":"Binding assays with Robo proteins, collagen gel repulsion/attraction assays, growth cone collapse assay using engineered truncated and mutant Slit2 constructs","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with purified fragments, mutagenesis of cleavage site, multiple orthogonal functional assays","pmids":["11404413"],"is_preprint":false},{"year":2006,"finding":"Slit2-induced growth cone collapse in Xenopus retinal neurons requires local protein synthesis and endocytosis; Slit2 activates MAP kinases and translation regulators in growth cones, and causes a protein-synthesis-dependent decrease in F-actin and increase in the actin-depolymerizing protein cofilin. Disruption of heparan sulfate blocks Slit2-induced protein synthesis and repulsion.","method":"Pharmacological inhibition of translation, endocytosis, MAPKs, and heparan sulfate; immunostaining for cofilin and F-actin in Xenopus retinal growth cones treated with Slit2-conditioned medium","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal pharmacological perturbations with quantitative readouts, replicated across conditions","pmids":["16423696"],"is_preprint":false},{"year":2007,"finding":"Slit2 inhibits CXCL12-induced chemotaxis and transendothelial migration of T cells and monocytes. CXCR4 physically associates with Robo1, and Slit2 treatment enhances this association. The CC3 motif of Robo1 mediates interaction with CXCR4. Slit2 blocks CXCL12-induced activation of Src and Lck kinases, Akt phosphorylation, and Rac activation, but not MAPK activation.","method":"Co-immunoprecipitation of CXCR4 and Robo1; chemotaxis and transendothelial migration assays; Western blot for kinase activation; domain deletion mutants of Robo1","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of CXCR4/Robo1, multiple signaling readouts, single lab","pmids":["17565045"],"is_preprint":false},{"year":2008,"finding":"Slit-2 mediates tumor-suppressive effects in breast cancer cells through coordinated regulation of the β-catenin and PI3K signaling pathways and by enhancing β-catenin/E-cadherin-mediated cell-cell adhesion.","method":"Slit2 overexpression in MCF-7 cells; in vitro proliferation and migration assays; in vivo tumor growth in mouse models; Western blot for β-catenin and PI3K pathway components","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function in vitro and in vivo with pathway analysis by Western blot, single lab","pmids":["18611862"],"is_preprint":false},{"year":2009,"finding":"Slit2 promotes vascular stability through Robo4 by forming a Robo4-paxillin complex at the cell surface that blocks activation of Arf6 and consequently Rac, via recruitment of Arf-GAP GIT1. This inhibits cellular protrusive activity underlying neovascularization and vascular leak.","method":"Co-immunoprecipitation of Robo4 with paxillin/Hic-5; biochemical assays for Arf6 and Rac activation; Arf6 inhibition in vivo (choroidal/retinal neovascularization models); cell biological protrusion assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted protein complex by Co-IP, in vitro signaling assays, and in vivo phenocopy with multiple orthogonal methods","pmids":["19855388"],"is_preprint":false},{"year":2009,"finding":"Slit2 inhibits glioma cell migration and invasion through Robo1-dependent suppression of Cdc42 activity. siRNA knockdown of Robo1 prevents Slit2-mediated Cdc42 inactivation and rescues cell migration inhibition.","method":"Cdc42 activity assay (GTP pull-down), Robo1 siRNA knockdown, modified Boyden chamber migration assay, in vivo intracranial tumor model","journal":"Neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi epistasis, biochemical GTPase assay, in vivo confirmation, single lab","pmids":["20008733"],"is_preprint":false},{"year":2009,"finding":"Slit2 crystal structure of the fourth domain (D4) reveals homodimerization using conserved residues on its concave face; D4 also binds heparan sulphate. Slit2 D4 causes growth cone collapse in chick retinal ganglion cells, which can be inhibited by exogenously added heparan sulphate.","method":"X-ray crystallography of Slit2 D4; heparan sulphate binding assay; chick retinal ganglion cell growth cone collapse assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation in growth cone assay and heparan sulphate competition","pmids":["19498462"],"is_preprint":false},{"year":2009,"finding":"Slit2 inhibits directional migration (chemotaxis) but not random movement of neutrophils toward fMLP, C5a, and IL-8, acting through Robo1. Mechanistically, Slit2 suppresses chemoattractant-induced actin barbed-end formation, cell polarization, and activation of Cdc42 and Rac2, without impairing major kinase pathways. In vivo, Slit2 reduces neutrophil recruitment in mouse peritoneal inflammation models.","method":"Video-microscopic live cell tracking; actin barbed-end formation assay; Cdc42/Rac2 activation assays; mouse peritoneal inflammation models; Robo1 expression confirmed by flow cytometry","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro mechanistic assays plus in vivo validation across three inflammation models","pmids":["19759280"],"is_preprint":false},{"year":2010,"finding":"Slit2 differentially regulates eosinophil and neutrophil chemotaxis through differential expression of srGAP1. In eosinophils (low srGAP1), Slit2/Robo1 activates Cdc42 and recruits PI3K to Robo1, enhancing eotaxin-induced chemotaxis. In neutrophils (high srGAP1), srGAP1 binding to Robo1 leads to Cdc42 inactivation and repulsion. Aerosol Slit2 accelerates eosinophil infiltration, while IV Slit2 reduces it.","method":"Measurement of srGAP1 expression in eosinophils vs neutrophils; PI3K recruitment to Robo1 by co-immunoprecipitation; Cdc42 activity assays; in vivo aerosol/IV Slit2 administration in OVA-sensitized mice; siRNA blockade of srGAP1","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, GTPase assays, in vivo experiments, single lab","pmids":["20944010"],"is_preprint":false},{"year":2011,"finding":"Slit-2 repels olfactory ensheathing cell (OEC) migration via Robo receptor. The repulsion requires Ca2+ release from internal stores through IP3 receptor channels. Slit-2-induced collapse of the leading front requires Ca2+-dependent cofilin activation (F-actin severing), while soma reversal requires spatial reversal of RhoA activity across the cell.","method":"Live cell imaging of OEC migration; Ca2+ imaging; pharmacological inhibition of IP3 receptors, cofilin, and RhoA/ROCK; immunostaining of cofilin activation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging plus multiple pharmacological epistasis experiments, single lab","pmids":["21187345"],"is_preprint":false},{"year":2012,"finding":"Slit2 is a potent inhibitor of platelet function. Robo1 is expressed on human and murine platelets and megakaryocytes. Slit2 impairs platelet adhesion and spreading by suppressing Akt activation, prevents platelet activation by ADP, prolongs bleeding times in vivo, delays vessel occlusion after arterial injury, and prevents stable occlusive thrombus formation.","method":"Immunofluorescence microscopy and flow cytometry for Robo1; static and shear microfluidic adhesion assays; Akt activation assays; murine tail bleeding and intravital microscopy models of arterial injury","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro and in vivo assays with mechanistic Akt readout, confirmed in two arterial injury models","pmids":["22865890"],"is_preprint":false},{"year":2012,"finding":"Slit2 and Robo3 directly modulate migration of GnRH neurons. Mice lacking Slit2 or Robo3 have a reduced number of GnRH neurons reaching the forebrain but normal complement of their axonal substrates, indicating a direct role on the neurons rather than via axonal patterning.","method":"Genetic knockout mouse models for Slit2 and Robo3; quantification of GnRH neuron numbers and axon patterns","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic loss-of-function with specific cellular phenotype, single lab","pmids":["22912413"],"is_preprint":false},{"year":2013,"finding":"Slit2-Robo4 signaling represses LPS-induced endothelial inflammation (cytokine secretion, ICAM-1 upregulation, monocyte adhesion) by inhibiting the Pyk2-NF-κB pathway downstream of TLR4. Robo1 has proinflammatory properties and is downregulated by Slit2 via miR-218.","method":"siRNA knockdown of Robo1 and Robo4 in HUVECs; cytokine/ICAM-1 measurement; NF-κB and Pyk2 phosphorylation assays; monocyte adhesion assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor-specific siRNA knockdown with signaling pathway readouts, single lab","pmids":["24272999"],"is_preprint":false},{"year":2014,"finding":"SLIT2 inhibits directed motility of pancreatic ductal adenocarcinoma cells through autocrine/paracrine ROBO1-mediated signaling. Restoring SLIT2 expression inhibits neural invasion and PDAC cell navigation along neurites; ROBO1 silencing in SLIT2-competent cells stimulates motility. Conditioned medium from SLIT2-competent PDAC cells impairs endothelial migration, but an N-terminal SLIT2 cleavage fragment stimulates it.","method":"SLIT2 restoration by transfection; ROBO1 siRNA knockdown; neural co-culture invasion assay; migration assays; in vivo tumor model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple gain- and loss-of-function experiments with specific phenotypic readouts, single lab","pmids":["24448236"],"is_preprint":false},{"year":2014,"finding":"Fibroblasts from healthy human tissues secrete Slit2, which inhibits monocyte-to-fibrocyte differentiation. In mice, injections of Slit2 inhibit bleomycin-induced lung fibrosis.","method":"Slit2 identification in fibroblast conditioned medium; fibrocyte differentiation assays with recombinant Slit2; in vivo bleomycin fibrosis model with Slit2 injections","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro functional assay with recombinant protein plus in vivo model, single lab","pmids":["25489114"],"is_preprint":false},{"year":2015,"finding":"Slit2 signals through Robo1 and Robo2 to promote retinal angiogenesis in vivo. Slit2/Robo1/Robo2 signaling promotes endothelial cell migration and is required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation.","method":"Conditional knockout mice for Slit2, Robo1, Robo2 (individual and combined); postnatal retinal vascular analysis; ocular neovascular disease model; Rac1 activation and lamellipodia assays in endothelial cells","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple conditional knockouts, mechanistic Rac1/lamellipodia readouts, in vivo disease model","pmids":["25894826"],"is_preprint":false},{"year":2015,"finding":"SLIT2/ROBO1 signaling regulates mammary stem cell self-renewal by governing the choice between asymmetric and symmetric cell divisions through the transcription factor SNAI1 and the spindle orientation protein mINSC. Loss of SLIT2/ROBO1 signaling increases nuclear SNAI1, which upregulates mInsc and shifts cap cells toward symmetric divisions, expanding the stem cell pool.","method":"Slit2/Robo1 knockout mouse mammary gland analysis; SNAI1 overexpression; mInsc expression measurement; cell division orientation quantification; rescue by SLIT2 treatment","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with mechanistic epistasis through SNAI1 and mInsc, single lab","pmids":["26440891"],"is_preprint":false},{"year":2015,"finding":"Slit2 serves as a β-catenin (Ctnnb1)-dependent retrograde signal from muscle to direct presynaptic (motoneuron) differentiation at the neuromuscular junction. Muscle-specific Ctnnb1 mutant lacking the transactivation domain fails to rescue presynaptic deficits. Transgenic Slit2 expression in muscle rescues presynaptic deficits caused by Ctnnb1 mutation. Slit2 immobilized on beads induces synaptophysin puncta in spinal cord axons.","method":"In vivo transgenic rescue experiments; bead-immobilized Slit2 applied to spinal cord explants; NMJ morphological analysis","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic rescue and bead-immobilization functional assay, single lab","pmids":["26159615"],"is_preprint":false},{"year":2015,"finding":"Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway: Slit2/Robo1 activates Src signaling, which downregulates E-cadherin, thereby activating Wnt/β-catenin.","method":"Slit2 transgenic mice crossed with ApcMin/+ and DMH/DSS colorectal carcinoma models; Western blot for Src, E-cadherin, β-catenin; in vitro signaling assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vivo mouse models with mechanistic pathway analysis, single lab","pmids":["25605242"],"is_preprint":false},{"year":2015,"finding":"CAF-secreted SLIT2 increases neurite outgrowth from dorsal root ganglia neurons and Schwann cell migration/proliferation through modulation of N-cadherin/β-catenin signaling. SLIT2/ROBO inhibition disrupts the stromal/neural connection.","method":"Co-culture of CAFs with DRG neurons; neurite outgrowth measurement; Schwann cell migration/proliferation assays; SLIT2/ROBO signaling inhibition; N-cadherin/β-catenin Western blot","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro functional assays with pathway analysis, single lab","pmids":["25590802"],"is_preprint":false},{"year":2016,"finding":"Full-length Slit2 is cleaved to generate smaller fragments in adipose tissue and in mice. The C-terminal fragment (Slit2-C, ~50 kDa) is the active thermogenic moiety that promotes adipose thermogenesis and augments energy expenditure. Mechanistically, Slit2 induces robust PKA signaling activation, which is required for its prothermogenic activity.","method":"Proteomics identification of Slit2 fragments; in vivo administration of Slit2-C fragment; PKA signaling assays; pharmacological inhibition of PKA; metabolic phenotyping in mice","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 1 / Strong — fragment identification with multiple in vivo functional assays and mechanistic PKA pathway validation","pmids":["26876562"],"is_preprint":false},{"year":2016,"finding":"SLIT2/ROBO2 signaling inhibits nonmuscle myosin IIA (NMIIA) activity and destabilizes kidney podocyte adhesion through SRGAP1. The myosin II regulatory light chain (MRLC) directly interacts with SRGAP1, and SLIT2 treatment promotes formation of a ROBO2/SRGAP1/NMIIA complex. SLIT2 stimulation decreases focal adhesion formation and reduces podocyte attachment to collagen. Podocyte-specific Robo2 knockout protects mice from hypertension-induced podocyte detachment.","method":"Co-immunoprecipitation of MRLC with SRGAP1 and ROBO2; NMIIA activity assays; focal adhesion staining; collagen adhesion assays; podocyte-specific Robo2 conditional knockout mice","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct protein interaction by Co-IP, in vitro biochemical assays, and in vivo genetic validation in conditional knockout","pmids":["27882344"],"is_preprint":false},{"year":2018,"finding":"Slit2/Robo1 signaling promotes liver fibrosis through activation of hepatic stellate cells (HSCs). In HSCs, Robo1 neutralizing antibody and Robo1 siRNA downregulate phosphorylation of Smad2, Smad3, PI3K, and AKT independently of TGF-β1, and inhibit α-SMA expression.","method":"Primary mouse HSCs and LX-2 human HSC line treated with Slit2/Robo1 manipulations; Western blot for Smad2/3, PI3K, AKT; Slit2 transgenic and Robo1/2+/- mice with CCl4-induced fibrosis","journal":"Journal of hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro signaling assays with siRNA and neutralizing antibody plus in vivo transgenic/heterozygous models, single lab","pmids":["26264936"],"is_preprint":false},{"year":2018,"finding":"USP33 deubiquitinates and stabilizes Robo1, thereby enhancing Slit2-Robo1 inhibitory signaling in gastric cancer cells. Co-IP analysis confirmed the interaction between Robo1 and USP33; ubiquitination analysis showed USP33 reduces Robo1 ubiquitination.","method":"Co-immunoprecipitation of Robo1 and USP33; ubiquitination assay; migration and EMT assays after USP33 manipulation","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with functional migration readouts, single lab","pmids":["30896071"],"is_preprint":false},{"year":2018,"finding":"CAPN5 directly cleaves SLIT2 in photoreceptors in vitro to generate SLIT2-N and SLIT2-C fragments. CAPN5 interacts with SLIT2 in retinal photoreceptors by co-immunoprecipitation. Increased CAPN5-mediated SLIT2 cleavage and secretion promotes PKA activation and proliferation in retinal pigment epithelium cells.","method":"In vitro cleavage assay (CAPN5 + SLIT2); co-immunoprecipitation in 661W cells; conditioned medium experiments; PKA activation by immunofluorescence and Western blot; CAPN5 knock-in mouse","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro cleavage reconstitution with Co-IP and downstream PKA signaling assay, single lab","pmids":["29610848"],"is_preprint":false},{"year":2018,"finding":"SLIT2 (expressed by CD34- orbital fibroblasts) attenuates the inflammatory phenotype of CD34+ fibrocytes in Graves' disease orbit. Conditioned medium from CD34- fibroblasts reduces MHC-2, thyroglobulin, TSHR, and AIRE expression in fibrocytes; depleting the medium of Slit2 reverses these effects. Recombinant human Slit2 mimics these effects. TSH induces Slit2 expression in GD-OF by enhancing both gene transcription and mRNA stability.","method":"Conditioned medium experiments with Slit2 depletion by antibody; recombinant Slit2 treatment; gene expression analysis; TSH stimulation experiments","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — depletion rescue experiment plus recombinant protein mimicry, single lab","pmids":["29752312"],"is_preprint":false},{"year":2018,"finding":"Direct negative cross-talk exists between SLIT2 and BMP-Gremlin signaling pathways: the SLIT2-Gremlin interaction inhibits both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity. BMP2 downregulates SLIT2 expression and promoter activity through canonical BMP/SMAD signaling; Gremlin treatment, BMP receptor inhibition, and SMAD4 knockdown rescue this repression.","method":"SLIT2-Gremlin binding assay; SLIT2-ROBO2 neuronal signaling assay; BMP activity assay in myoblasts; SLIT2 promoter-reporter assay; SMAD4 siRNA knockdown; BMP2 treatment of nephron progenitor cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays including binding, promoter, and signaling in multiple cell types, single lab","pmids":["29317497"],"is_preprint":false},{"year":2019,"finding":"SLIT2 inhibits osteoclastogenesis and bone resorption through ROBO1-mediated suppression of Cdc42 activity. Binding ELISA confirmed SLIT2 directly binds ROBO1 (not ROBO3) in bone marrow-derived macrophages. Robo1 siRNA blocks SLIT2-mediated suppression of osteoclastogenesis. Cdc42 overexpression reverses SLIT2-mediated inhibition.","method":"Binding ELISA for SLIT2-ROBO1 interaction; Cdc42 activity assay; Robo1 siRNA knockdown; osteoclast differentiation and bone resorption assays; Cdc42 overexpression rescue","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay, GTPase epistasis, receptor-specific siRNA, single lab","pmids":["31084928"],"is_preprint":false},{"year":2020,"finding":"Tumor cell-derived double-stranded RNA activates TLR3 in endothelial cells to induce SLIT2 expression. Endothelial-derived SLIT2 and its receptor ROBO1 on tumor cells promote cancer cell migration toward endothelial cells and intravasation. Deleting endothelial Slit2 suppresses metastasis, while deleting tumoural Slit2 enhances it, demonstrating that the same gene can promote or suppress cancer depending on its cellular source.","method":"Genetically tagged endothelial ribosome profiling; conditional endothelial and tumor-specific Slit2 deletion in mouse breast/lung cancer models; TLR3 activation studies; in vitro migration assays; endogenous retroviral element RNA detection","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — compartment-specific conditional knockouts, mechanistic TLR3 upstream identification, replicated in two cancer models","pmids":["32999457"],"is_preprint":false},{"year":2020,"finding":"SLIT2 inhibits macropinocytosis in macrophages and RAS-transformed cancer cells by inducing cytoskeletal changes via Roundabout receptors. In vivo, SLIT2 attenuates muramyl dipeptide uptake, preventing NOD2-dependent NF-κB activation and CXCL1 secretion. Blocking endogenous SLIT2 enhances CXCL1 secretion.","method":"Macropinocytosis assays in macrophages and RAS-transformed cells; in vivo SLIT2 administration; NOD2/NF-κB pathway readouts; endogenous SLIT2 blocking experiments; CXCL1 measurement","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro and in vivo functional assays with gain and loss of function plus downstream signaling pathway validation","pmids":["32807784"],"is_preprint":false},{"year":2021,"finding":"SLIT2 promotes microglia/macrophage chemotaxis and tumor-supportive polarization in glioblastoma via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimic SLIT2 knockdown effects on tumor growth and the tumor microenvironment.","method":"SLIT2 knockdown in mouse glioma cells; macrophage-specific Robo1/Robo2 deletion; PI3K-γ activation assays; SLIT2 trap systemic delivery; tumor growth and immune profiling","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — receptor-specific genetic knockouts plus systemic inhibitor with mechanistic PI3K-γ readout, multiple orthogonal approaches","pmids":["34181595"],"is_preprint":false},{"year":2021,"finding":"Slit2 reduces breast cancer metastasis by activating M1-like tumor-associated macrophages (TAMs) and enhancing their phagocytosis of tumor cells via suppression of IL-6. Slit2 also diminishes tumor fibrosis by increasing matrix metalloproteinase 13 expression in M1-TAMs.","method":"Slit2 overexpression in syngeneic and xenograft mammary tumor models; in vitro and in vivo phagocytosis assays; IL-6 manipulation; MMP13 expression analysis; macrophage polarization assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vivo models with mechanistic IL-6 and MMP13 pathway identification, single lab","pmids":["34400395"],"is_preprint":false},{"year":2022,"finding":"IGF2BP3 (an m6A reader) promotes TNBC metastasis by binding to and destabilizing m6A-methylated SLIT2 mRNA, thereby impairing SLIT2/ROBO1 downstream signaling and activating PI3K/AKT and MEK/ERK pathways.","method":"RNA m6A modification analysis; IGF2BP3 binding to SLIT2 mRNA; mRNA stability assays; pathway activation by Western blot; in vivo xenograft metastasis model","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA binding and stability assays with downstream signaling pathway readouts plus in vivo validation, single lab","pmids":["36250924"],"is_preprint":false},{"year":2023,"finding":"CAF-derived SLIT2 promotes gastric cancer metastasis by binding ROBO1, which interacts with serine/threonine kinase NEK9 (identified by mass spectrometry). SLIT2 enhances ROBO1-NEK9 binding. NEK9 kinase domain interacts with ROBO1 intracellular domain. NEK9 directly phosphorylates TRIM28 and cortactin (CTTN). TRIM28 acts as a transcriptional elongation factor and, together with STAT3 and NF-κB p100, drives CTTN transcription, inducing cytoskeletal reorganization.","method":"Mass spectrometry identification of ROBO1 interactors; Co-IP of ROBO1 and NEK9; domain deletion analysis; in vitro kinase assay for NEK9 phosphorylation of TRIM28 and CTTN; transcriptional assays; in vitro and in vivo metastasis models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification, Co-IP, kinase assay, and transcriptional analysis in single lab","pmids":["37443302"],"is_preprint":false},{"year":2023,"finding":"SLIT2-ROBO1 axis facilitates liver metastasis of PDAC through 'coadaptation' dependent on the dependence receptor (DR) characteristics of ROBO1. A neutralizing antibody targeting ROBO1 significantly attenuates PDAC liver metastasis by preventing the coadaptation effect.","method":"Mouse PDAC metastasis model with CT/3D bioluminescence imaging; ROBO1 dependence receptor characterization; ROBO1-neutralizing antibody treatment","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo model with ROBO1 DR characterization and antibody intervention, single lab","pmids":["36792623"],"is_preprint":false},{"year":2016,"finding":"ISL1-based LIM complexes (ISL1-LHX3 and ISL1-LHX4) bind to the Slit2 enhancer and drive endogenous Slit2 expression in somatic motor neurons and branchiomotor neurons. Loss of Isl1 results in near-complete absence of Slit2 transcript in brainstem motor neurons and causes cranial motor neuron positioning and axon guidance defects.","method":"ChIP-seq analysis showing ISL1 binding to Slit2 enhancer; Isl1 conditional knockout mice; in situ hybridization and immunostaining for Slit2; luciferase reporter assay for Slit2 enhancer","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP binding, genetic loss-of-function, and reporter assay, single lab","pmids":["27819291"],"is_preprint":false},{"year":2004,"finding":"EphrinA5 and slit2 both induce growth cone collapse in chick retinal ganglion cells through shared pathways (PI3K and Src family kinases) but diverge in that non-classical PKC isoforms are required for ephrinA5-mediated but not slit2-mediated collapse. PI3K and Src family kinases are co-purified by affinity chromatography with the slit2 receptor Roundabout.","method":"Pharmacological inhibition of PI3K, Src, and PKC in chick retinal ganglion cell growth cone collapse assay; affinity chromatography of Roundabout with PI3K; colocalization of Src and Roundabout","journal":"Journal of neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity chromatography, pharmacological epistasis, colocalization, single lab","pmids":["15007828"],"is_preprint":false},{"year":2020,"finding":"NADPH oxidase 2 (Nox2) acts downstream of slit2/Robo2 in retinal ganglion cell axon guidance. Slit2-mediated growth cone collapse, axonal retraction, and repulsive turning are eliminated by Nox inhibition. Slit2 treatment increases growth cone hydrogen peroxide levels via Nox2 activation. In vivo, astray/nox2 double heterozygotes show decreased retinotectal innervation, suggesting genetic interaction.","method":"Pharmacological Nox inhibition; ROS biosensor imaging in growth cones; Nox2 mutant zebrafish; double heterozygote genetic interaction analysis; in vitro growth cone collapse and turning assays","journal":"Developmental neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic epistasis with ROS biosensor imaging, single lab","pmids":["33191581"],"is_preprint":false},{"year":2015,"finding":"Slit2 promotes angiogenesis in endothelial cells via the Robo1-VEGFR2-ERK1/2 pathway. Slit2 overexpression upregulates Robo1, VEGFR2, and phosphorylated ERK1/2 in HUVECs and in laser-treated transgenic mouse retina/choroid, promoting endothelial proliferation, migration, and tube formation.","method":"Slit2 overexpression in HUVECs; laser-induced CNV in Slit2 transgenic mice; Western blot for VEGFR2 and p-ERK1/2; proliferation, migration, tube formation assays","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo gain-of-function with pathway readout, single lab","pmids":["26244297"],"is_preprint":false},{"year":2018,"finding":"Slit2/Robo1 signaling promotes angiogenesis in glomerular endothelial cells under diabetic conditions through the PI3K/Akt/VEGF pathway. Robo1 siRNA suppresses PI3K/Akt and VEGF activation; PI3K inhibitor blocks VEGF but not Robo1 expression, placing Robo1 upstream of PI3K/Akt.","method":"Slit2 siRNA in mesangial cells; conditioned medium on glomerular endothelial cells; Robo1 siRNA; PI3K inhibitor; Western blot for PI3K/Akt/VEGF/HIF-1α; proliferation, migration, tube formation assays","journal":"Angiogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor siRNA epistasis with signaling inhibitors and functional assays, single lab","pmids":["29299781"],"is_preprint":false},{"year":2020,"finding":"Slit2-Robo1 signaling promotes cardiac fibrosis through crosstalk with the TGF-β1/Smad pathway. Recombinant Slit2 activates TGF-β1/Smad signaling in cardiac fibroblasts and upregulates collagen I and periostin; Slit2 siRNA downregulates Smad2/3 and collagen I.","method":"Recombinant Slit2 treatment of cardiac fibroblasts; Slit2 siRNA and TGF-β1 siRNA; Western blot for Smad2/3, collagen I, PI3K/Akt; TAC mouse model; Robo1 antagonist R5","journal":"ESC heart failure","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA epistasis with signaling readouts and in vivo TAC model, single lab","pmids":["33236535"],"is_preprint":false},{"year":2022,"finding":"CCN2/CTGF directly interacts with the 7th-9th EGF repeats and C-terminal cysteine knot motif of Slit2 in hepatic stellate cells and fibrotic livers. CCN2/CTGF and Slit2 synergistically activate PI3K/AKT in primary HSCs, and soluble Robo1-Fc chimera inhibits these activities.","method":"Co-immunoprecipitation and domain mapping of CCN2/CTGF with Slit2; PI3K/AKT activation assays; Robo1-Fc inhibition; Ccn2/Ctgf conditional knockout mice with CCl4 fibrosis","journal":"Journal of cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction with domain mapping plus functional signaling readout, single lab","pmids":["36469291"],"is_preprint":false}],"current_model":"SLIT2 is a large secreted glycoprotein that is proteolytically cleaved into N-terminal (Slit2-N) and C-terminal (Slit2-C) fragments; Slit2-N binds Robo1/Robo2 receptors to mediate chemorepulsion and growth cone collapse (requiring local protein synthesis, cofilin activation, Cdc42/Rac inhibition via srGAP1, and PI3K/Src kinases), while Slit2-C acts independently through PKA to promote adipose thermogenesis; Robo4-mediated vascular stability involves a Robo4-paxillin-GIT1-Arf6-Rac axis, ROBO2 signals through SRGAP1 to inhibit nonmuscle myosin IIA and reduce podocyte adhesion, and the pathway broadly suppresses leukocyte chemotaxis, platelet activation, and macropinocytosis through Rho-family GTPase regulation, while its role in angiogenesis and cancer depends on cellular context and the specific Robo receptor expressed."},"narrative":{"mechanistic_narrative":"SLIT2 is a large secreted glycoprotein that functions as a guidance and motility-regulating cue, originally defined as a chemorepellent that collapses olfactory and hippocampal axon growth cones through the Robo1 and Robo2 receptors in the developing forebrain [PMID:10197527]. It is proteolytically processed in vivo into a Robo-binding N-terminal fragment (Slit2-N) and a C-terminal fragment (Slit2-C); only Slit2-N and the uncleaved form bind Robo and drive repulsion and growth-cone collapse, whereas the uncleaved form antagonizes Slit2-N-induced axon branching, establishing that fragment identity dictates output [PMID:11404413]. Heparan sulfate is an obligatory cofactor, and the structurally dimerizing D4 domain both binds heparan sulfate and is sufficient to collapse growth cones [PMID:19498462, PMID:16423696]. Growth-cone repulsion proceeds through local protein synthesis, endocytosis, MAPK activation, and cofilin-mediated F-actin depolymerization [PMID:16423696], converging on a core mechanism in which Slit2/Robo signaling reorganizes the actin cytoskeleton by controlling Rho-family GTPases—suppressing Cdc42 and Rac in repulsive contexts via srGAP1, with PI3K and Src kinases physically associated with Robo [PMID:15007828, PMID:19759280, PMID:31084928]. The same Rho-GTPase logic underlies SLIT2's role as a broad suppressor of directed cell motility and inflammation: it blocks chemoattractant-driven chemotaxis of neutrophils, T cells and monocytes by inhibiting Cdc42/Rac2 and CXCR4-coupled signaling [PMID:19759280, PMID:17565045], with cell-type-specific outcomes set by srGAP1 levels [PMID:20944010]. Through Robo4, SLIT2 stabilizes vasculature by assembling a Robo4-paxillin complex that recruits GIT1 to block Arf6 and Rac, limiting endothelial protrusion and inflammation [PMID:19855388, PMID:24272999], and it inhibits platelet adhesion via suppression of Akt [PMID:22865890] and restrains macropinocytosis to dampen NOD2/NF-κB inflammatory signaling [PMID:32807784]. SLIT2 also acts in tissue homeostasis and disease beyond neurons: Slit2-C drives PKA-dependent adipose thermogenesis [PMID:26876562], SLIT2/ROBO2 destabilizes podocyte adhesion by inhibiting nonmuscle myosin IIA through SRGAP1 [PMID:27882344], and SLIT2/ROBO1 controls mammary stem-cell division orientation via SNAI1/mINSC [PMID:26440891]. In angiogenesis and cancer its effects are strongly context- and receptor-dependent: it can promote endothelial migration and Rac1-dependent angiogenesis through Robo1/Robo2 [PMID:25894826, PMID:26244297], while acting as a tumor suppressor of migration and invasion in glioma and pancreatic cancer through Robo1-mediated Cdc42 suppression [PMID:20008733, PMID:24448236], with the net pro- versus anti-metastatic outcome determined by the cellular source of SLIT2 [PMID:32999457].","teleology":[{"year":1999,"claim":"Established SLIT2's foundational function by showing the human protein directly repels axons and collapses growth cones through Robo receptors, defining the Slit-Robo guidance axis in mammals.","evidence":"Collagen gel repulsion and growth cone collapse assays with recombinant human Slit2 on olfactory and hippocampal axons","pmids":["10197527"],"confidence":"High","gaps":["Did not resolve downstream cytoskeletal effectors","Receptor binding inferred functionally, not structurally"]},{"year":2001,"claim":"Resolved that SLIT2 is proteolytically processed and that Robo binding and distinct activities partition between fragments, showing fragment identity determines repulsion versus branching outputs.","evidence":"Robo binding assays and functional repulsion/branching assays with engineered truncated and cleavage-site mutant Slit2","pmids":["11404413"],"confidence":"High","gaps":["Protease responsible for in vivo cleavage not identified","Mechanism of full-length antagonism unresolved"]},{"year":2006,"claim":"Defined the downstream machinery of growth-cone repulsion, showing it requires local protein synthesis, endocytosis, MAPK activation, and cofilin-driven actin depolymerization, and depends on heparan sulfate.","evidence":"Pharmacological perturbation and cofilin/F-actin immunostaining in Xenopus retinal growth cones","pmids":["16423696"],"confidence":"High","gaps":["Identity of locally synthesized proteins not defined","Link from MAPK to cofilin not mapped"]},{"year":2009,"claim":"Provided structural and biochemical basis for SLIT2 dimerization and heparan-sulfate dependence, with the D4 domain sufficient to collapse growth cones.","evidence":"X-ray crystallography of Slit2 D4, heparan sulfate binding, and chick retinal ganglion cell collapse assay","pmids":["19498462"],"confidence":"High","gaps":["No structure of the Slit2-Robo complex","Functional role of dimerization in vivo untested"]},{"year":2009,"claim":"Extended SLIT2 function beyond neurons by showing it suppresses leukocyte chemotaxis through Robo1-dependent inhibition of Cdc42/Rac without blocking major kinase pathways, establishing an anti-inflammatory role.","evidence":"Live-cell tracking, actin barbed-end and GTPase activation assays, and mouse peritonitis models","pmids":["19759280"],"confidence":"High","gaps":["srGAP coupling to Robo1 not directly shown here","Selectivity for directed versus random motility mechanism incomplete"]},{"year":2009,"claim":"Identified a Robo4-specific vascular-stabilizing mechanism, showing SLIT2 assembles a Robo4-paxillin complex that recruits GIT1 to block Arf6 and Rac, distinct from Robo1/2 neuronal signaling.","evidence":"Co-IP of Robo4 with paxillin, Arf6/Rac activation assays, and in vivo neovascularization models","pmids":["19855388"],"confidence":"High","gaps":["How Robo4 selects this complex over Robo1/2 outputs unclear","Direct GAP activity of GIT1 on Arf6 in this context inferred"]},{"year":2010,"claim":"Showed cell-context determines SLIT2 output, with srGAP1 abundance switching Robo1 signaling between Cdc42 activation (eosinophils) and inactivation (neutrophils).","evidence":"srGAP1 expression profiling, PI3K-Robo1 Co-IP, Cdc42 assays, and in vivo airway eosinophil models","pmids":["20944010"],"confidence":"Medium","gaps":["Single lab","Quantitative srGAP1 threshold not defined"]},{"year":2012,"claim":"Demonstrated SLIT2 directly inhibits platelet function, broadening its anti-adhesive role to hemostasis via Akt suppression.","evidence":"Robo1 detection, microfluidic adhesion and Akt assays, and murine bleeding/arterial-injury models","pmids":["22865890"],"confidence":"High","gaps":["Upstream link from Robo1 to Akt not mapped","Which Slit2 fragment is active not tested"]},{"year":2016,"claim":"Uncovered a fragment- and receptor-independent metabolic function, showing Slit2-C drives PKA-dependent adipose thermogenesis distinct from Robo-mediated repulsion.","evidence":"Proteomic fragment identification, in vivo Slit2-C administration, and PKA inhibition with metabolic phenotyping","pmids":["26876562"],"confidence":"High","gaps":["Receptor/coupling for Slit2-C-PKA signaling unidentified","Protease generating Slit2-C in adipose tissue undefined"]},{"year":2016,"claim":"Defined a renal mechanism in which SLIT2/ROBO2 destabilizes podocyte adhesion by forming a ROBO2/SRGAP1/NMIIA complex that inhibits nonmuscle myosin IIA.","evidence":"Co-IP of MRLC with SRGAP1/ROBO2, focal adhesion and adhesion assays, and podocyte-specific Robo2 knockout mice","pmids":["27882344"],"confidence":"High","gaps":["How SRGAP1 connects to NMIIA enzymatically not fully resolved","Relevance to human kidney disease inferred from mouse"]},{"year":2015,"claim":"Showed SLIT2/Robo1/Robo2 can promote rather than restrain angiogenesis, being required for Slit2- and VEGF-induced Rac1 activation and endothelial lamellipodia, demonstrating context-dependent vascular outputs.","evidence":"Conditional Slit2/Robo1/Robo2 knockouts, retinal vascular analysis, and Rac1/lamellipodia assays","pmids":["25894826"],"confidence":"High","gaps":["Reconciliation with Robo4 anti-angiogenic role incomplete","Direct VEGFR crosstalk mechanism not defined here"]},{"year":2020,"claim":"Resolved the apparent paradox of SLIT2 in cancer by showing its pro- versus anti-metastatic effect depends on cellular source, with endothelial SLIT2 promoting and tumoral SLIT2 suppressing metastasis.","evidence":"Compartment-specific conditional Slit2 deletion in breast/lung cancer models and TLR3-driven endothelial induction","pmids":["32999457"],"confidence":"High","gaps":["Why identical ligand yields opposite outcomes by source mechanistically incomplete","Receptor expression differences across compartments not fully dissected"]},{"year":2020,"claim":"Identified SLIT2 as an inhibitor of macropinocytosis that limits innate inflammatory signaling, linking cytoskeletal control to NOD2/NF-κB suppression.","evidence":"Macropinocytosis assays in macrophages and RAS-transformed cells, in vivo administration, and CXCL1/NF-κB readouts","pmids":["32807784"],"confidence":"High","gaps":["Which Robo receptor mediates the effect not pinned down","Direct cytoskeletal effector for macropinosome closure unspecified"]},{"year":2023,"claim":"Began mapping intracellular Robo1 signaling complexes in cancer, identifying a SLIT2-enhanced ROBO1-NEK9 axis that phosphorylates TRIM28 and cortactin to drive cytoskeletal remodeling.","evidence":"Mass spectrometry of ROBO1 interactors, Co-IP, in vitro kinase and transcriptional assays, and metastasis models","pmids":["37443302"],"confidence":"Medium","gaps":["Single lab","Generality of NEK9 coupling beyond gastric cancer untested"]},{"year":null,"claim":"How the choice between SLIT2's opposing outputs—repulsion versus attraction, pro- versus anti-angiogenic, tumor-suppressive versus metastasis-promoting—is encoded by fragment identity, receptor (Robo1/2/3/4) repertoire, srGAP1 levels, and cellular source remains unresolved at a unifying mechanistic level.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model linking fragment processing to receptor selection","Protease(s) governing in vivo cleavage context incompletely defined","Structural basis of receptor-specific signaling complexes unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1,16,28]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,8,22]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[2,7]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[1,15,21,26,29]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[22,42]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,8,22]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,12,16,18]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,8,13,30]}],"complexes":["Robo4-paxillin-GIT1 complex","ROBO2/SRGAP1/NMIIA complex"],"partners":["ROBO1","ROBO2","ROBO4","SRGAP1","CXCR4","USP33","NEK9","CCN2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O94813","full_name":"Slit homolog 2 protein","aliases":[],"length_aa":1529,"mass_kda":169.9,"function":"Thought to act as molecular guidance cue in cellular migration, and function appears to be mediated by interaction with roundabout homolog receptors. During neural development involved in axonal navigation at the ventral midline of the neural tube and projection of axons to different regions. SLIT1 and SLIT2 seem to be essential for midline guidance in the forebrain by acting as repulsive signal preventing inappropriate midline crossing by axons projecting from the olfactory bulb. In spinal cord development may play a role in guiding commissural axons once they reached the floor plate by modulating the response to netrin. In vitro, silences the attractive effect of NTN1 but not its growth-stimulatory effect and silencing requires the formation of a ROBO1-DCC complex. May be implicated in spinal cord midline post-crossing axon repulsion. In vitro, only commissural axons that crossed the midline responded to SLIT2. In the developing visual system appears to function as repellent for retinal ganglion axons by providing a repulsion that directs these axons along their appropriate paths prior to, and after passage through, the optic chiasm. In vitro, collapses and repels retinal ganglion cell growth cones. Seems to play a role in branching and arborization of CNS sensory axons, and in neuronal cell migration. In vitro, Slit homolog 2 protein N-product, but not Slit homolog 2 protein C-product, repels olfactory bulb (OB) but not dorsal root ganglia (DRG) axons, induces OB growth cones collapse and induces branching of DRG axons. Seems to be involved in regulating leukocyte migration","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/O94813/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SLIT2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SLIT2","total_profiled":1310},"omim":[{"mim_id":"616771","title":"MICRO RNA 218-2; MIR218-2","url":"https://www.omim.org/entry/616771"},{"mim_id":"616770","title":"MICRO RNA 218-1; MIR218-1","url":"https://www.omim.org/entry/616770"},{"mim_id":"612759","title":"SYNESTHESIA","url":"https://www.omim.org/entry/612759"},{"mim_id":"610937","title":"RPGRIP1-LIKE; RPGRIP1L","url":"https://www.omim.org/entry/610937"},{"mim_id":"610878","title":"VESICOURETERAL REFLUX 2; VUR2","url":"https://www.omim.org/entry/610878"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SLIT2"},"hgnc":{"alias_symbol":["Slit-2"],"prev_symbol":["SLIL3"]},"alphafold":{"accession":"O94813","domains":[{"cath_id":"3.80.10.10","chopping":"276-478","consensus_level":"medium","plddt":88.8439,"start":276,"end":478},{"cath_id":"3.80.10.10","chopping":"507-666","consensus_level":"medium","plddt":89.4792,"start":507,"end":666},{"cath_id":"2.10.25.10","chopping":"922-958","consensus_level":"medium","plddt":79.4892,"start":922,"end":958},{"cath_id":"2.10.25.10","chopping":"1088-1114","consensus_level":"medium","plddt":75.9178,"start":1088,"end":1114},{"cath_id":"2.60.120.200","chopping":"1152-1285_1303-1363","consensus_level":"medium","plddt":76.699,"start":1152,"end":1363},{"cath_id":"2.10.25.10","chopping":"1416-1451","consensus_level":"medium","plddt":74.1678,"start":1416,"end":1451},{"cath_id":"2.10.90","chopping":"1455-1527","consensus_level":"high","plddt":83.1729,"start":1455,"end":1527}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94813","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94813-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94813-F1-predicted_aligned_error_v6.png","plddt_mean":79.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SLIT2","jax_strain_url":"https://www.jax.org/strain/search?query=SLIT2"},"sequence":{"accession":"O94813","fasta_url":"https://rest.uniprot.org/uniprotkb/O94813.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94813/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94813"}},"corpus_meta":[{"pmid":"21385904","id":"PMC_21385904","title":"MiR-218 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\"confidence_rationale\": \"Tier 1 / Strong — direct in vitro functional assay with recombinant protein, replicated across multiple axon types, consistent with Drosophila genetic data\",\n      \"pmids\": [\"10197527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Slit2 is proteolytically cleaved in vivo into a 140 kDa N-terminal fragment (Slit2-N) and a 55-60 kDa C-terminal fragment (Slit2-C). Only Slit2-N (and uncleaved Slit2-U) bind Robo receptors. Slit2-N repels olfactory bulb axons and induces growth cone collapse, while only Slit2-N (not Slit2-C) induces DRG axon branching. The full-length uncleaved form antagonizes branching by Slit2-N.\",\n      \"method\": \"Binding assays with Robo proteins, collagen gel repulsion/attraction assays, growth cone collapse assay using engineered truncated and mutant Slit2 constructs\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with purified fragments, mutagenesis of cleavage site, multiple orthogonal functional assays\",\n      \"pmids\": [\"11404413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Slit2-induced growth cone collapse in Xenopus retinal neurons requires local protein synthesis and endocytosis; Slit2 activates MAP kinases and translation regulators in growth cones, and causes a protein-synthesis-dependent decrease in F-actin and increase in the actin-depolymerizing protein cofilin. Disruption of heparan sulfate blocks Slit2-induced protein synthesis and repulsion.\",\n      \"method\": \"Pharmacological inhibition of translation, endocytosis, MAPKs, and heparan sulfate; immunostaining for cofilin and F-actin in Xenopus retinal growth cones treated with Slit2-conditioned medium\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal pharmacological perturbations with quantitative readouts, replicated across conditions\",\n      \"pmids\": [\"16423696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Slit2 inhibits CXCL12-induced chemotaxis and transendothelial migration of T cells and monocytes. CXCR4 physically associates with Robo1, and Slit2 treatment enhances this association. The CC3 motif of Robo1 mediates interaction with CXCR4. Slit2 blocks CXCL12-induced activation of Src and Lck kinases, Akt phosphorylation, and Rac activation, but not MAPK activation.\",\n      \"method\": \"Co-immunoprecipitation of CXCR4 and Robo1; chemotaxis and transendothelial migration assays; Western blot for kinase activation; domain deletion mutants of Robo1\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of CXCR4/Robo1, multiple signaling readouts, single lab\",\n      \"pmids\": [\"17565045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Slit-2 mediates tumor-suppressive effects in breast cancer cells through coordinated regulation of the β-catenin and PI3K signaling pathways and by enhancing β-catenin/E-cadherin-mediated cell-cell adhesion.\",\n      \"method\": \"Slit2 overexpression in MCF-7 cells; in vitro proliferation and migration assays; in vivo tumor growth in mouse models; Western blot for β-catenin and PI3K pathway components\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function in vitro and in vivo with pathway analysis by Western blot, single lab\",\n      \"pmids\": [\"18611862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Slit2 promotes vascular stability through Robo4 by forming a Robo4-paxillin complex at the cell surface that blocks activation of Arf6 and consequently Rac, via recruitment of Arf-GAP GIT1. This inhibits cellular protrusive activity underlying neovascularization and vascular leak.\",\n      \"method\": \"Co-immunoprecipitation of Robo4 with paxillin/Hic-5; biochemical assays for Arf6 and Rac activation; Arf6 inhibition in vivo (choroidal/retinal neovascularization models); cell biological protrusion assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted protein complex by Co-IP, in vitro signaling assays, and in vivo phenocopy with multiple orthogonal methods\",\n      \"pmids\": [\"19855388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Slit2 inhibits glioma cell migration and invasion through Robo1-dependent suppression of Cdc42 activity. siRNA knockdown of Robo1 prevents Slit2-mediated Cdc42 inactivation and rescues cell migration inhibition.\",\n      \"method\": \"Cdc42 activity assay (GTP pull-down), Robo1 siRNA knockdown, modified Boyden chamber migration assay, in vivo intracranial tumor model\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi epistasis, biochemical GTPase assay, in vivo confirmation, single lab\",\n      \"pmids\": [\"20008733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Slit2 crystal structure of the fourth domain (D4) reveals homodimerization using conserved residues on its concave face; D4 also binds heparan sulphate. Slit2 D4 causes growth cone collapse in chick retinal ganglion cells, which can be inhibited by exogenously added heparan sulphate.\",\n      \"method\": \"X-ray crystallography of Slit2 D4; heparan sulphate binding assay; chick retinal ganglion cell growth cone collapse assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation in growth cone assay and heparan sulphate competition\",\n      \"pmids\": [\"19498462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Slit2 inhibits directional migration (chemotaxis) but not random movement of neutrophils toward fMLP, C5a, and IL-8, acting through Robo1. Mechanistically, Slit2 suppresses chemoattractant-induced actin barbed-end formation, cell polarization, and activation of Cdc42 and Rac2, without impairing major kinase pathways. In vivo, Slit2 reduces neutrophil recruitment in mouse peritoneal inflammation models.\",\n      \"method\": \"Video-microscopic live cell tracking; actin barbed-end formation assay; Cdc42/Rac2 activation assays; mouse peritoneal inflammation models; Robo1 expression confirmed by flow cytometry\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro mechanistic assays plus in vivo validation across three inflammation models\",\n      \"pmids\": [\"19759280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Slit2 differentially regulates eosinophil and neutrophil chemotaxis through differential expression of srGAP1. In eosinophils (low srGAP1), Slit2/Robo1 activates Cdc42 and recruits PI3K to Robo1, enhancing eotaxin-induced chemotaxis. In neutrophils (high srGAP1), srGAP1 binding to Robo1 leads to Cdc42 inactivation and repulsion. Aerosol Slit2 accelerates eosinophil infiltration, while IV Slit2 reduces it.\",\n      \"method\": \"Measurement of srGAP1 expression in eosinophils vs neutrophils; PI3K recruitment to Robo1 by co-immunoprecipitation; Cdc42 activity assays; in vivo aerosol/IV Slit2 administration in OVA-sensitized mice; siRNA blockade of srGAP1\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, GTPase assays, in vivo experiments, single lab\",\n      \"pmids\": [\"20944010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Slit-2 repels olfactory ensheathing cell (OEC) migration via Robo receptor. The repulsion requires Ca2+ release from internal stores through IP3 receptor channels. Slit-2-induced collapse of the leading front requires Ca2+-dependent cofilin activation (F-actin severing), while soma reversal requires spatial reversal of RhoA activity across the cell.\",\n      \"method\": \"Live cell imaging of OEC migration; Ca2+ imaging; pharmacological inhibition of IP3 receptors, cofilin, and RhoA/ROCK; immunostaining of cofilin activation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging plus multiple pharmacological epistasis experiments, single lab\",\n      \"pmids\": [\"21187345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Slit2 is a potent inhibitor of platelet function. Robo1 is expressed on human and murine platelets and megakaryocytes. Slit2 impairs platelet adhesion and spreading by suppressing Akt activation, prevents platelet activation by ADP, prolongs bleeding times in vivo, delays vessel occlusion after arterial injury, and prevents stable occlusive thrombus formation.\",\n      \"method\": \"Immunofluorescence microscopy and flow cytometry for Robo1; static and shear microfluidic adhesion assays; Akt activation assays; murine tail bleeding and intravital microscopy models of arterial injury\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro and in vivo assays with mechanistic Akt readout, confirmed in two arterial injury models\",\n      \"pmids\": [\"22865890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Slit2 and Robo3 directly modulate migration of GnRH neurons. Mice lacking Slit2 or Robo3 have a reduced number of GnRH neurons reaching the forebrain but normal complement of their axonal substrates, indicating a direct role on the neurons rather than via axonal patterning.\",\n      \"method\": \"Genetic knockout mouse models for Slit2 and Robo3; quantification of GnRH neuron numbers and axon patterns\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic loss-of-function with specific cellular phenotype, single lab\",\n      \"pmids\": [\"22912413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Slit2-Robo4 signaling represses LPS-induced endothelial inflammation (cytokine secretion, ICAM-1 upregulation, monocyte adhesion) by inhibiting the Pyk2-NF-κB pathway downstream of TLR4. Robo1 has proinflammatory properties and is downregulated by Slit2 via miR-218.\",\n      \"method\": \"siRNA knockdown of Robo1 and Robo4 in HUVECs; cytokine/ICAM-1 measurement; NF-κB and Pyk2 phosphorylation assays; monocyte adhesion assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor-specific siRNA knockdown with signaling pathway readouts, single lab\",\n      \"pmids\": [\"24272999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SLIT2 inhibits directed motility of pancreatic ductal adenocarcinoma cells through autocrine/paracrine ROBO1-mediated signaling. Restoring SLIT2 expression inhibits neural invasion and PDAC cell navigation along neurites; ROBO1 silencing in SLIT2-competent cells stimulates motility. Conditioned medium from SLIT2-competent PDAC cells impairs endothelial migration, but an N-terminal SLIT2 cleavage fragment stimulates it.\",\n      \"method\": \"SLIT2 restoration by transfection; ROBO1 siRNA knockdown; neural co-culture invasion assay; migration assays; in vivo tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple gain- and loss-of-function experiments with specific phenotypic readouts, single lab\",\n      \"pmids\": [\"24448236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Fibroblasts from healthy human tissues secrete Slit2, which inhibits monocyte-to-fibrocyte differentiation. In mice, injections of Slit2 inhibit bleomycin-induced lung fibrosis.\",\n      \"method\": \"Slit2 identification in fibroblast conditioned medium; fibrocyte differentiation assays with recombinant Slit2; in vivo bleomycin fibrosis model with Slit2 injections\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro functional assay with recombinant protein plus in vivo model, single lab\",\n      \"pmids\": [\"25489114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2 signals through Robo1 and Robo2 to promote retinal angiogenesis in vivo. Slit2/Robo1/Robo2 signaling promotes endothelial cell migration and is required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation.\",\n      \"method\": \"Conditional knockout mice for Slit2, Robo1, Robo2 (individual and combined); postnatal retinal vascular analysis; ocular neovascular disease model; Rac1 activation and lamellipodia assays in endothelial cells\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple conditional knockouts, mechanistic Rac1/lamellipodia readouts, in vivo disease model\",\n      \"pmids\": [\"25894826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SLIT2/ROBO1 signaling regulates mammary stem cell self-renewal by governing the choice between asymmetric and symmetric cell divisions through the transcription factor SNAI1 and the spindle orientation protein mINSC. Loss of SLIT2/ROBO1 signaling increases nuclear SNAI1, which upregulates mInsc and shifts cap cells toward symmetric divisions, expanding the stem cell pool.\",\n      \"method\": \"Slit2/Robo1 knockout mouse mammary gland analysis; SNAI1 overexpression; mInsc expression measurement; cell division orientation quantification; rescue by SLIT2 treatment\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with mechanistic epistasis through SNAI1 and mInsc, single lab\",\n      \"pmids\": [\"26440891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2 serves as a β-catenin (Ctnnb1)-dependent retrograde signal from muscle to direct presynaptic (motoneuron) differentiation at the neuromuscular junction. Muscle-specific Ctnnb1 mutant lacking the transactivation domain fails to rescue presynaptic deficits. Transgenic Slit2 expression in muscle rescues presynaptic deficits caused by Ctnnb1 mutation. Slit2 immobilized on beads induces synaptophysin puncta in spinal cord axons.\",\n      \"method\": \"In vivo transgenic rescue experiments; bead-immobilized Slit2 applied to spinal cord explants; NMJ morphological analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic rescue and bead-immobilization functional assay, single lab\",\n      \"pmids\": [\"26159615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway: Slit2/Robo1 activates Src signaling, which downregulates E-cadherin, thereby activating Wnt/β-catenin.\",\n      \"method\": \"Slit2 transgenic mice crossed with ApcMin/+ and DMH/DSS colorectal carcinoma models; Western blot for Src, E-cadherin, β-catenin; in vitro signaling assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vivo mouse models with mechanistic pathway analysis, single lab\",\n      \"pmids\": [\"25605242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CAF-secreted SLIT2 increases neurite outgrowth from dorsal root ganglia neurons and Schwann cell migration/proliferation through modulation of N-cadherin/β-catenin signaling. SLIT2/ROBO inhibition disrupts the stromal/neural connection.\",\n      \"method\": \"Co-culture of CAFs with DRG neurons; neurite outgrowth measurement; Schwann cell migration/proliferation assays; SLIT2/ROBO signaling inhibition; N-cadherin/β-catenin Western blot\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro functional assays with pathway analysis, single lab\",\n      \"pmids\": [\"25590802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Full-length Slit2 is cleaved to generate smaller fragments in adipose tissue and in mice. The C-terminal fragment (Slit2-C, ~50 kDa) is the active thermogenic moiety that promotes adipose thermogenesis and augments energy expenditure. Mechanistically, Slit2 induces robust PKA signaling activation, which is required for its prothermogenic activity.\",\n      \"method\": \"Proteomics identification of Slit2 fragments; in vivo administration of Slit2-C fragment; PKA signaling assays; pharmacological inhibition of PKA; metabolic phenotyping in mice\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — fragment identification with multiple in vivo functional assays and mechanistic PKA pathway validation\",\n      \"pmids\": [\"26876562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SLIT2/ROBO2 signaling inhibits nonmuscle myosin IIA (NMIIA) activity and destabilizes kidney podocyte adhesion through SRGAP1. The myosin II regulatory light chain (MRLC) directly interacts with SRGAP1, and SLIT2 treatment promotes formation of a ROBO2/SRGAP1/NMIIA complex. SLIT2 stimulation decreases focal adhesion formation and reduces podocyte attachment to collagen. Podocyte-specific Robo2 knockout protects mice from hypertension-induced podocyte detachment.\",\n      \"method\": \"Co-immunoprecipitation of MRLC with SRGAP1 and ROBO2; NMIIA activity assays; focal adhesion staining; collagen adhesion assays; podocyte-specific Robo2 conditional knockout mice\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct protein interaction by Co-IP, in vitro biochemical assays, and in vivo genetic validation in conditional knockout\",\n      \"pmids\": [\"27882344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Slit2/Robo1 signaling promotes liver fibrosis through activation of hepatic stellate cells (HSCs). In HSCs, Robo1 neutralizing antibody and Robo1 siRNA downregulate phosphorylation of Smad2, Smad3, PI3K, and AKT independently of TGF-β1, and inhibit α-SMA expression.\",\n      \"method\": \"Primary mouse HSCs and LX-2 human HSC line treated with Slit2/Robo1 manipulations; Western blot for Smad2/3, PI3K, AKT; Slit2 transgenic and Robo1/2+/- mice with CCl4-induced fibrosis\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro signaling assays with siRNA and neutralizing antibody plus in vivo transgenic/heterozygous models, single lab\",\n      \"pmids\": [\"26264936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP33 deubiquitinates and stabilizes Robo1, thereby enhancing Slit2-Robo1 inhibitory signaling in gastric cancer cells. Co-IP analysis confirmed the interaction between Robo1 and USP33; ubiquitination analysis showed USP33 reduces Robo1 ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation of Robo1 and USP33; ubiquitination assay; migration and EMT assays after USP33 manipulation\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with functional migration readouts, single lab\",\n      \"pmids\": [\"30896071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CAPN5 directly cleaves SLIT2 in photoreceptors in vitro to generate SLIT2-N and SLIT2-C fragments. CAPN5 interacts with SLIT2 in retinal photoreceptors by co-immunoprecipitation. Increased CAPN5-mediated SLIT2 cleavage and secretion promotes PKA activation and proliferation in retinal pigment epithelium cells.\",\n      \"method\": \"In vitro cleavage assay (CAPN5 + SLIT2); co-immunoprecipitation in 661W cells; conditioned medium experiments; PKA activation by immunofluorescence and Western blot; CAPN5 knock-in mouse\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro cleavage reconstitution with Co-IP and downstream PKA signaling assay, single lab\",\n      \"pmids\": [\"29610848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SLIT2 (expressed by CD34- orbital fibroblasts) attenuates the inflammatory phenotype of CD34+ fibrocytes in Graves' disease orbit. Conditioned medium from CD34- fibroblasts reduces MHC-2, thyroglobulin, TSHR, and AIRE expression in fibrocytes; depleting the medium of Slit2 reverses these effects. Recombinant human Slit2 mimics these effects. TSH induces Slit2 expression in GD-OF by enhancing both gene transcription and mRNA stability.\",\n      \"method\": \"Conditioned medium experiments with Slit2 depletion by antibody; recombinant Slit2 treatment; gene expression analysis; TSH stimulation experiments\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion rescue experiment plus recombinant protein mimicry, single lab\",\n      \"pmids\": [\"29752312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Direct negative cross-talk exists between SLIT2 and BMP-Gremlin signaling pathways: the SLIT2-Gremlin interaction inhibits both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity. BMP2 downregulates SLIT2 expression and promoter activity through canonical BMP/SMAD signaling; Gremlin treatment, BMP receptor inhibition, and SMAD4 knockdown rescue this repression.\",\n      \"method\": \"SLIT2-Gremlin binding assay; SLIT2-ROBO2 neuronal signaling assay; BMP activity assay in myoblasts; SLIT2 promoter-reporter assay; SMAD4 siRNA knockdown; BMP2 treatment of nephron progenitor cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays including binding, promoter, and signaling in multiple cell types, single lab\",\n      \"pmids\": [\"29317497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SLIT2 inhibits osteoclastogenesis and bone resorption through ROBO1-mediated suppression of Cdc42 activity. Binding ELISA confirmed SLIT2 directly binds ROBO1 (not ROBO3) in bone marrow-derived macrophages. Robo1 siRNA blocks SLIT2-mediated suppression of osteoclastogenesis. Cdc42 overexpression reverses SLIT2-mediated inhibition.\",\n      \"method\": \"Binding ELISA for SLIT2-ROBO1 interaction; Cdc42 activity assay; Robo1 siRNA knockdown; osteoclast differentiation and bone resorption assays; Cdc42 overexpression rescue\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay, GTPase epistasis, receptor-specific siRNA, single lab\",\n      \"pmids\": [\"31084928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tumor cell-derived double-stranded RNA activates TLR3 in endothelial cells to induce SLIT2 expression. Endothelial-derived SLIT2 and its receptor ROBO1 on tumor cells promote cancer cell migration toward endothelial cells and intravasation. Deleting endothelial Slit2 suppresses metastasis, while deleting tumoural Slit2 enhances it, demonstrating that the same gene can promote or suppress cancer depending on its cellular source.\",\n      \"method\": \"Genetically tagged endothelial ribosome profiling; conditional endothelial and tumor-specific Slit2 deletion in mouse breast/lung cancer models; TLR3 activation studies; in vitro migration assays; endogenous retroviral element RNA detection\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — compartment-specific conditional knockouts, mechanistic TLR3 upstream identification, replicated in two cancer models\",\n      \"pmids\": [\"32999457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SLIT2 inhibits macropinocytosis in macrophages and RAS-transformed cancer cells by inducing cytoskeletal changes via Roundabout receptors. In vivo, SLIT2 attenuates muramyl dipeptide uptake, preventing NOD2-dependent NF-κB activation and CXCL1 secretion. Blocking endogenous SLIT2 enhances CXCL1 secretion.\",\n      \"method\": \"Macropinocytosis assays in macrophages and RAS-transformed cells; in vivo SLIT2 administration; NOD2/NF-κB pathway readouts; endogenous SLIT2 blocking experiments; CXCL1 measurement\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro and in vivo functional assays with gain and loss of function plus downstream signaling pathway validation\",\n      \"pmids\": [\"32807784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SLIT2 promotes microglia/macrophage chemotaxis and tumor-supportive polarization in glioblastoma via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimic SLIT2 knockdown effects on tumor growth and the tumor microenvironment.\",\n      \"method\": \"SLIT2 knockdown in mouse glioma cells; macrophage-specific Robo1/Robo2 deletion; PI3K-γ activation assays; SLIT2 trap systemic delivery; tumor growth and immune profiling\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — receptor-specific genetic knockouts plus systemic inhibitor with mechanistic PI3K-γ readout, multiple orthogonal approaches\",\n      \"pmids\": [\"34181595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Slit2 reduces breast cancer metastasis by activating M1-like tumor-associated macrophages (TAMs) and enhancing their phagocytosis of tumor cells via suppression of IL-6. Slit2 also diminishes tumor fibrosis by increasing matrix metalloproteinase 13 expression in M1-TAMs.\",\n      \"method\": \"Slit2 overexpression in syngeneic and xenograft mammary tumor models; in vitro and in vivo phagocytosis assays; IL-6 manipulation; MMP13 expression analysis; macrophage polarization assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vivo models with mechanistic IL-6 and MMP13 pathway identification, single lab\",\n      \"pmids\": [\"34400395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"IGF2BP3 (an m6A reader) promotes TNBC metastasis by binding to and destabilizing m6A-methylated SLIT2 mRNA, thereby impairing SLIT2/ROBO1 downstream signaling and activating PI3K/AKT and MEK/ERK pathways.\",\n      \"method\": \"RNA m6A modification analysis; IGF2BP3 binding to SLIT2 mRNA; mRNA stability assays; pathway activation by Western blot; in vivo xenograft metastasis model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA binding and stability assays with downstream signaling pathway readouts plus in vivo validation, single lab\",\n      \"pmids\": [\"36250924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CAF-derived SLIT2 promotes gastric cancer metastasis by binding ROBO1, which interacts with serine/threonine kinase NEK9 (identified by mass spectrometry). SLIT2 enhances ROBO1-NEK9 binding. NEK9 kinase domain interacts with ROBO1 intracellular domain. NEK9 directly phosphorylates TRIM28 and cortactin (CTTN). TRIM28 acts as a transcriptional elongation factor and, together with STAT3 and NF-κB p100, drives CTTN transcription, inducing cytoskeletal reorganization.\",\n      \"method\": \"Mass spectrometry identification of ROBO1 interactors; Co-IP of ROBO1 and NEK9; domain deletion analysis; in vitro kinase assay for NEK9 phosphorylation of TRIM28 and CTTN; transcriptional assays; in vitro and in vivo metastasis models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification, Co-IP, kinase assay, and transcriptional analysis in single lab\",\n      \"pmids\": [\"37443302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SLIT2-ROBO1 axis facilitates liver metastasis of PDAC through 'coadaptation' dependent on the dependence receptor (DR) characteristics of ROBO1. A neutralizing antibody targeting ROBO1 significantly attenuates PDAC liver metastasis by preventing the coadaptation effect.\",\n      \"method\": \"Mouse PDAC metastasis model with CT/3D bioluminescence imaging; ROBO1 dependence receptor characterization; ROBO1-neutralizing antibody treatment\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo model with ROBO1 DR characterization and antibody intervention, single lab\",\n      \"pmids\": [\"36792623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ISL1-based LIM complexes (ISL1-LHX3 and ISL1-LHX4) bind to the Slit2 enhancer and drive endogenous Slit2 expression in somatic motor neurons and branchiomotor neurons. Loss of Isl1 results in near-complete absence of Slit2 transcript in brainstem motor neurons and causes cranial motor neuron positioning and axon guidance defects.\",\n      \"method\": \"ChIP-seq analysis showing ISL1 binding to Slit2 enhancer; Isl1 conditional knockout mice; in situ hybridization and immunostaining for Slit2; luciferase reporter assay for Slit2 enhancer\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP binding, genetic loss-of-function, and reporter assay, single lab\",\n      \"pmids\": [\"27819291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EphrinA5 and slit2 both induce growth cone collapse in chick retinal ganglion cells through shared pathways (PI3K and Src family kinases) but diverge in that non-classical PKC isoforms are required for ephrinA5-mediated but not slit2-mediated collapse. PI3K and Src family kinases are co-purified by affinity chromatography with the slit2 receptor Roundabout.\",\n      \"method\": \"Pharmacological inhibition of PI3K, Src, and PKC in chick retinal ganglion cell growth cone collapse assay; affinity chromatography of Roundabout with PI3K; colocalization of Src and Roundabout\",\n      \"journal\": \"Journal of neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity chromatography, pharmacological epistasis, colocalization, single lab\",\n      \"pmids\": [\"15007828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NADPH oxidase 2 (Nox2) acts downstream of slit2/Robo2 in retinal ganglion cell axon guidance. Slit2-mediated growth cone collapse, axonal retraction, and repulsive turning are eliminated by Nox inhibition. Slit2 treatment increases growth cone hydrogen peroxide levels via Nox2 activation. In vivo, astray/nox2 double heterozygotes show decreased retinotectal innervation, suggesting genetic interaction.\",\n      \"method\": \"Pharmacological Nox inhibition; ROS biosensor imaging in growth cones; Nox2 mutant zebrafish; double heterozygote genetic interaction analysis; in vitro growth cone collapse and turning assays\",\n      \"journal\": \"Developmental neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic epistasis with ROS biosensor imaging, single lab\",\n      \"pmids\": [\"33191581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2 promotes angiogenesis in endothelial cells via the Robo1-VEGFR2-ERK1/2 pathway. Slit2 overexpression upregulates Robo1, VEGFR2, and phosphorylated ERK1/2 in HUVECs and in laser-treated transgenic mouse retina/choroid, promoting endothelial proliferation, migration, and tube formation.\",\n      \"method\": \"Slit2 overexpression in HUVECs; laser-induced CNV in Slit2 transgenic mice; Western blot for VEGFR2 and p-ERK1/2; proliferation, migration, tube formation assays\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo gain-of-function with pathway readout, single lab\",\n      \"pmids\": [\"26244297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Slit2/Robo1 signaling promotes angiogenesis in glomerular endothelial cells under diabetic conditions through the PI3K/Akt/VEGF pathway. Robo1 siRNA suppresses PI3K/Akt and VEGF activation; PI3K inhibitor blocks VEGF but not Robo1 expression, placing Robo1 upstream of PI3K/Akt.\",\n      \"method\": \"Slit2 siRNA in mesangial cells; conditioned medium on glomerular endothelial cells; Robo1 siRNA; PI3K inhibitor; Western blot for PI3K/Akt/VEGF/HIF-1α; proliferation, migration, tube formation assays\",\n      \"journal\": \"Angiogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor siRNA epistasis with signaling inhibitors and functional assays, single lab\",\n      \"pmids\": [\"29299781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Slit2-Robo1 signaling promotes cardiac fibrosis through crosstalk with the TGF-β1/Smad pathway. Recombinant Slit2 activates TGF-β1/Smad signaling in cardiac fibroblasts and upregulates collagen I and periostin; Slit2 siRNA downregulates Smad2/3 and collagen I.\",\n      \"method\": \"Recombinant Slit2 treatment of cardiac fibroblasts; Slit2 siRNA and TGF-β1 siRNA; Western blot for Smad2/3, collagen I, PI3K/Akt; TAC mouse model; Robo1 antagonist R5\",\n      \"journal\": \"ESC heart failure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA epistasis with signaling readouts and in vivo TAC model, single lab\",\n      \"pmids\": [\"33236535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CCN2/CTGF directly interacts with the 7th-9th EGF repeats and C-terminal cysteine knot motif of Slit2 in hepatic stellate cells and fibrotic livers. CCN2/CTGF and Slit2 synergistically activate PI3K/AKT in primary HSCs, and soluble Robo1-Fc chimera inhibits these activities.\",\n      \"method\": \"Co-immunoprecipitation and domain mapping of CCN2/CTGF with Slit2; PI3K/AKT activation assays; Robo1-Fc inhibition; Ccn2/Ctgf conditional knockout mice with CCl4 fibrosis\",\n      \"journal\": \"Journal of cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction with domain mapping plus functional signaling readout, single lab\",\n      \"pmids\": [\"36469291\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SLIT2 is a large secreted glycoprotein that is proteolytically cleaved into N-terminal (Slit2-N) and C-terminal (Slit2-C) fragments; Slit2-N binds Robo1/Robo2 receptors to mediate chemorepulsion and growth cone collapse (requiring local protein synthesis, cofilin activation, Cdc42/Rac inhibition via srGAP1, and PI3K/Src kinases), while Slit2-C acts independently through PKA to promote adipose thermogenesis; Robo4-mediated vascular stability involves a Robo4-paxillin-GIT1-Arf6-Rac axis, ROBO2 signals through SRGAP1 to inhibit nonmuscle myosin IIA and reduce podocyte adhesion, and the pathway broadly suppresses leukocyte chemotaxis, platelet activation, and macropinocytosis through Rho-family GTPase regulation, while its role in angiogenesis and cancer depends on cellular context and the specific Robo receptor expressed.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SLIT2 is a large secreted glycoprotein that functions as a guidance and motility-regulating cue, originally defined as a chemorepellent that collapses olfactory and hippocampal axon growth cones through the Robo1 and Robo2 receptors in the developing forebrain [#0]. It is proteolytically processed in vivo into a Robo-binding N-terminal fragment (Slit2-N) and a C-terminal fragment (Slit2-C); only Slit2-N and the uncleaved form bind Robo and drive repulsion and growth-cone collapse, whereas the uncleaved form antagonizes Slit2-N-induced axon branching, establishing that fragment identity dictates output [#1]. Heparan sulfate is an obligatory cofactor, and the structurally dimerizing D4 domain both binds heparan sulfate and is sufficient to collapse growth cones [#7, #2]. Growth-cone repulsion proceeds through local protein synthesis, endocytosis, MAPK activation, and cofilin-mediated F-actin depolymerization [#2], converging on a core mechanism in which Slit2/Robo signaling reorganizes the actin cytoskeleton by controlling Rho-family GTPases—suppressing Cdc42 and Rac in repulsive contexts via srGAP1, with PI3K and Src kinases physically associated with Robo [#37, #8, #28]. The same Rho-GTPase logic underlies SLIT2's role as a broad suppressor of directed cell motility and inflammation: it blocks chemoattractant-driven chemotaxis of neutrophils, T cells and monocytes by inhibiting Cdc42/Rac2 and CXCR4-coupled signaling [#8, #3], with cell-type-specific outcomes set by srGAP1 levels [#9]. Through Robo4, SLIT2 stabilizes vasculature by assembling a Robo4-paxillin complex that recruits GIT1 to block Arf6 and Rac, limiting endothelial protrusion and inflammation [#5, #13], and it inhibits platelet adhesion via suppression of Akt [#11] and restrains macropinocytosis to dampen NOD2/NF-κB inflammatory signaling [#30]. SLIT2 also acts in tissue homeostasis and disease beyond neurons: Slit2-C drives PKA-dependent adipose thermogenesis [#21], SLIT2/ROBO2 destabilizes podocyte adhesion by inhibiting nonmuscle myosin IIA through SRGAP1 [#22], and SLIT2/ROBO1 controls mammary stem-cell division orientation via SNAI1/mINSC [#17]. In angiogenesis and cancer its effects are strongly context- and receptor-dependent: it can promote endothelial migration and Rac1-dependent angiogenesis through Robo1/Robo2 [#16, #39], while acting as a tumor suppressor of migration and invasion in glioma and pancreatic cancer through Robo1-mediated Cdc42 suppression [#6, #14], with the net pro- versus anti-metastatic outcome determined by the cellular source of SLIT2 [#29].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established SLIT2's foundational function by showing the human protein directly repels axons and collapses growth cones through Robo receptors, defining the Slit-Robo guidance axis in mammals.\",\n      \"evidence\": \"Collagen gel repulsion and growth cone collapse assays with recombinant human Slit2 on olfactory and hippocampal axons\",\n      \"pmids\": [\"10197527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve downstream cytoskeletal effectors\", \"Receptor binding inferred functionally, not structurally\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved that SLIT2 is proteolytically processed and that Robo binding and distinct activities partition between fragments, showing fragment identity determines repulsion versus branching outputs.\",\n      \"evidence\": \"Robo binding assays and functional repulsion/branching assays with engineered truncated and cleavage-site mutant Slit2\",\n      \"pmids\": [\"11404413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Protease responsible for in vivo cleavage not identified\", \"Mechanism of full-length antagonism unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the downstream machinery of growth-cone repulsion, showing it requires local protein synthesis, endocytosis, MAPK activation, and cofilin-driven actin depolymerization, and depends on heparan sulfate.\",\n      \"evidence\": \"Pharmacological perturbation and cofilin/F-actin immunostaining in Xenopus retinal growth cones\",\n      \"pmids\": [\"16423696\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of locally synthesized proteins not defined\", \"Link from MAPK to cofilin not mapped\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Provided structural and biochemical basis for SLIT2 dimerization and heparan-sulfate dependence, with the D4 domain sufficient to collapse growth cones.\",\n      \"evidence\": \"X-ray crystallography of Slit2 D4, heparan sulfate binding, and chick retinal ganglion cell collapse assay\",\n      \"pmids\": [\"19498462\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the Slit2-Robo complex\", \"Functional role of dimerization in vivo untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended SLIT2 function beyond neurons by showing it suppresses leukocyte chemotaxis through Robo1-dependent inhibition of Cdc42/Rac without blocking major kinase pathways, establishing an anti-inflammatory role.\",\n      \"evidence\": \"Live-cell tracking, actin barbed-end and GTPase activation assays, and mouse peritonitis models\",\n      \"pmids\": [\"19759280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"srGAP coupling to Robo1 not directly shown here\", \"Selectivity for directed versus random motility mechanism incomplete\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified a Robo4-specific vascular-stabilizing mechanism, showing SLIT2 assembles a Robo4-paxillin complex that recruits GIT1 to block Arf6 and Rac, distinct from Robo1/2 neuronal signaling.\",\n      \"evidence\": \"Co-IP of Robo4 with paxillin, Arf6/Rac activation assays, and in vivo neovascularization models\",\n      \"pmids\": [\"19855388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Robo4 selects this complex over Robo1/2 outputs unclear\", \"Direct GAP activity of GIT1 on Arf6 in this context inferred\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed cell-context determines SLIT2 output, with srGAP1 abundance switching Robo1 signaling between Cdc42 activation (eosinophils) and inactivation (neutrophils).\",\n      \"evidence\": \"srGAP1 expression profiling, PI3K-Robo1 Co-IP, Cdc42 assays, and in vivo airway eosinophil models\",\n      \"pmids\": [\"20944010\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Quantitative srGAP1 threshold not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated SLIT2 directly inhibits platelet function, broadening its anti-adhesive role to hemostasis via Akt suppression.\",\n      \"evidence\": \"Robo1 detection, microfluidic adhesion and Akt assays, and murine bleeding/arterial-injury models\",\n      \"pmids\": [\"22865890\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream link from Robo1 to Akt not mapped\", \"Which Slit2 fragment is active not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Uncovered a fragment- and receptor-independent metabolic function, showing Slit2-C drives PKA-dependent adipose thermogenesis distinct from Robo-mediated repulsion.\",\n      \"evidence\": \"Proteomic fragment identification, in vivo Slit2-C administration, and PKA inhibition with metabolic phenotyping\",\n      \"pmids\": [\"26876562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor/coupling for Slit2-C-PKA signaling unidentified\", \"Protease generating Slit2-C in adipose tissue undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined a renal mechanism in which SLIT2/ROBO2 destabilizes podocyte adhesion by forming a ROBO2/SRGAP1/NMIIA complex that inhibits nonmuscle myosin IIA.\",\n      \"evidence\": \"Co-IP of MRLC with SRGAP1/ROBO2, focal adhesion and adhesion assays, and podocyte-specific Robo2 knockout mice\",\n      \"pmids\": [\"27882344\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How SRGAP1 connects to NMIIA enzymatically not fully resolved\", \"Relevance to human kidney disease inferred from mouse\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed SLIT2/Robo1/Robo2 can promote rather than restrain angiogenesis, being required for Slit2- and VEGF-induced Rac1 activation and endothelial lamellipodia, demonstrating context-dependent vascular outputs.\",\n      \"evidence\": \"Conditional Slit2/Robo1/Robo2 knockouts, retinal vascular analysis, and Rac1/lamellipodia assays\",\n      \"pmids\": [\"25894826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with Robo4 anti-angiogenic role incomplete\", \"Direct VEGFR crosstalk mechanism not defined here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the apparent paradox of SLIT2 in cancer by showing its pro- versus anti-metastatic effect depends on cellular source, with endothelial SLIT2 promoting and tumoral SLIT2 suppressing metastasis.\",\n      \"evidence\": \"Compartment-specific conditional Slit2 deletion in breast/lung cancer models and TLR3-driven endothelial induction\",\n      \"pmids\": [\"32999457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why identical ligand yields opposite outcomes by source mechanistically incomplete\", \"Receptor expression differences across compartments not fully dissected\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified SLIT2 as an inhibitor of macropinocytosis that limits innate inflammatory signaling, linking cytoskeletal control to NOD2/NF-κB suppression.\",\n      \"evidence\": \"Macropinocytosis assays in macrophages and RAS-transformed cells, in vivo administration, and CXCL1/NF-κB readouts\",\n      \"pmids\": [\"32807784\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which Robo receptor mediates the effect not pinned down\", \"Direct cytoskeletal effector for macropinosome closure unspecified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Began mapping intracellular Robo1 signaling complexes in cancer, identifying a SLIT2-enhanced ROBO1-NEK9 axis that phosphorylates TRIM28 and cortactin to drive cytoskeletal remodeling.\",\n      \"evidence\": \"Mass spectrometry of ROBO1 interactors, Co-IP, in vitro kinase and transcriptional assays, and metastasis models\",\n      \"pmids\": [\"37443302\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Generality of NEK9 coupling beyond gastric cancer untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the choice between SLIT2's opposing outputs—repulsion versus attraction, pro- versus anti-angiogenic, tumor-suppressive versus metastasis-promoting—is encoded by fragment identity, receptor (Robo1/2/3/4) repertoire, srGAP1 levels, and cellular source remains unresolved at a unifying mechanistic level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model linking fragment processing to receptor selection\", \"Protease(s) governing in vivo cleavage context incompletely defined\", \"Structural basis of receptor-specific signaling complexes unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 16, 28]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 8, 22]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [1, 15, 21, 26, 29]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [22, 42]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 8, 22]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 12, 16, 18]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 8, 13, 30]}\n    ],\n    \"complexes\": [\n      \"Robo4-paxillin-GIT1 complex\",\n      \"ROBO2/SRGAP1/NMIIA complex\"\n    ],\n    \"partners\": [\n      \"ROBO1\",\n      \"ROBO2\",\n      \"ROBO4\",\n      \"SRGAP1\",\n      \"CXCR4\",\n      \"USP33\",\n      \"NEK9\",\n      \"CCN2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}