{"gene":"SEMA3G","run_date":"2026-04-28T20:42:07","timeline":{"discoveries":[{"year":2012,"finding":"SEMA3G overexpression in U251MG glioma cells inhibits cell migration and invasion, and reduces MMP2 activity, functioning through both cell-autonomous and paracrine mechanisms.","method":"Stable overexpression in glioma cells, conditioned media treatment, Matrigel invasion assay, MMP2 activity assay","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — clean overexpression/KD with defined cellular phenotype and mechanistic readout (MMP2 activity), single lab","pmids":["22562223"],"is_preprint":false},{"year":2015,"finding":"PPAR-γ directly binds the SEMA3G promoter and transcriptionally activates SEMA3G expression, which in turn promotes endothelial cell migration via neuropilin-2 (NRP2) as the receptor.","method":"Chromatin immunoprecipitation (ChIP), transient transfection reporter assay, siRNA knockdown, anti-NRP2 neutralizing antibody, HUVEC migration assay","journal":"Journal of cellular biochemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal ChIP + reporter assay + receptor blockade with functional rescue, multiple orthogonal methods in single study","pmids":["25335934"],"is_preprint":false},{"year":2020,"finding":"SEMA3G promotes adipocyte differentiation and lipogenesis; its knockdown inhibits high-fat diet-induced obesity. SEMA3G acts through PI3K/Akt/GSK3β signaling in adipose tissue and the AMPK/SREBP-1c pathway in liver.","method":"shRNA lentiviral knockdown in 3T3-L1 cells and primary preadipocytes, in vivo HFD mouse model, pathway inhibitor assays","journal":"The Journal of endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro and in vivo KD with defined metabolic phenotype and pathway identification, single lab","pmids":["31648186"],"is_preprint":false},{"year":2020,"finding":"A loss-of-function SEMA3G mutation alters binding to PlexinA receptors and attenuates migration of immortalized GnRH neurons; SEMA3G is expressed along the migratory route of GnRH neurons and in the developing pituitary, regulating GnRH neuron development.","method":"Homozygosity mapping, exome sequencing, homology modelling, cell migration assay in immortalized GnRH neurons, in vivo mouse expression analysis","journal":"Neuroendocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — in silico structural modeling combined with in vitro migration assay and in vivo expression, single lab with multiple orthogonal methods","pmids":["32365351"],"is_preprint":false},{"year":2023,"finding":"ECs-derived SEMA3G promotes vascular smooth muscle cell (VSMC) proliferation and migration via Nrp2/PlexinA1 receptors, mechanistically by inhibiting LATS1 and activating YAP, leading to upregulation of cyclin D1, cyclin E, MMP2, and MMP9.","method":"Recombinant SEMA3G treatment of HASMCs, receptor inhibition (anti-Nrp2/PlexinA1), YAP inhibitor (verteporfin), western blot, cell cycle analysis, in vivo diabetic mouse femoral injury model","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 — receptor-specific inhibition, pathway inhibitor rescue, in vivo validation, multiple orthogonal methods","pmids":["36720439"],"is_preprint":false},{"year":2023,"finding":"miR-146b-5p directly targets and suppresses SEMA3G expression in clear cell renal cell carcinoma; SEMA3G loss promotes EMT, cell migration, and invasion via Notch and TGF-β signaling pathways.","method":"Dual-luciferase reporter assay, qRT-PCR, western blot (EMT markers, Notch/TGF-β pathway proteins, MMP2/9), Transwell assay, wound healing assay","journal":"Cell division","confidence":"Medium","confidence_rationale":"Tier 2 — direct miRNA-target validation by dual-luciferase plus functional assays, single lab","pmids":["36882799"],"is_preprint":false},{"year":2024,"finding":"BMP9 transcriptionally upregulates SEMA3G via ALK1/Smad1/Smad5 canonical signaling and the transcription factor SOX17 in pulmonary endothelial cells; SEMA3G then inhibits VEGFR2 phosphorylation and VEGF-mediated endothelial migration and network formation.","method":"Knockdown studies (siRNA for BMPR2, ACTR2A, Smad1, Smad5, SOX17, SEMA3G), VEGFR2 phosphorylation assay, functional endothelial migration and network formation assays, recombinant SEMA3G treatment","journal":"Vascular pharmacology","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic knockdowns defining pathway hierarchy plus receptor phosphorylation assay and functional rescue with recombinant protein, multiple orthogonal methods","pmids":["38795838"],"is_preprint":false},{"year":2025,"finding":"SEMA3G acts as an immune checkpoint ligand binding NRP1 (with stronger affinity than NRP2) to suppress CD8+ T-cell cytotoxicity; CRISPR/Cas9 deletion or neutralizing antibody blockade of SEMA3G restores T-cell cytotoxicity and inhibits tumor growth in vivo.","method":"Receptor binding/affinity screening, CRISPR/Cas9 knockout, neutralizing antibody blockade, CD8+ T-cell cytotoxicity assay, in vivo tumor growth assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — receptor affinity determination combined with genetic KO and antibody blockade with in vitro and in vivo functional validation, multiple orthogonal methods","pmids":["39652581"],"is_preprint":false},{"year":2025,"finding":"ECs-derived SEMA3G activates NRP2/PLXNA1 on glioblastoma stem cells (GSCs) in a paracrine manner, inducing Cdc42 inactivation and dissociation of Cdc42 from WWP2; free WWP2 then ubiquitinates c-Myc, leading to c-Myc degradation and impairment of GSC stemness.","method":"Co-IP, genetic deletion of Sema3G in ECs, overexpression, recombinant SEMA3G protein treatment, ubiquitination assay, in vivo GBM mouse survival model","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical mechanism (ubiquitination assay, Co-IP dissociation of Cdc42/WWP2) combined with genetic KO and in vivo survival, rigorous mechanistic dissection","pmids":["40533501"],"is_preprint":false}],"current_model":"SEMA3G is a secreted class-3 semaphorin that signals through NRP1, NRP2, and PlexinA family receptors to regulate diverse cellular processes: it inhibits VEGFR2 phosphorylation downstream of BMP9/ALK1/Smad/SOX17 signaling to suppress angiogenesis; activates YAP via NRP2/PlexinA1 to promote vascular smooth muscle cell proliferation; induces c-Myc ubiquitination via a NRP2/PLXNA1–Cdc42–WWP2 cascade to suppress glioblastoma stem cell stemness; binds NRP1 on T cells to suppress cytotoxic immunity; regulates GnRH neuron migration through PlexinA receptors; and promotes adipogenesis via PI3K/Akt/GSK3β and AMPK/SREBP-1c pathways."},"narrative":{"teleology":[{"year":2012,"claim":"The initial question of whether SEMA3G has functional relevance in tumor cell behavior was addressed by showing that overexpression in glioma cells inhibits migration and invasion through both cell-autonomous and paracrine mechanisms involving MMP2 suppression, establishing SEMA3G as a negative regulator of tumor cell motility.","evidence":"Stable overexpression in U251MG glioma cells with conditioned media transfer and Matrigel invasion assays","pmids":["22562223"],"confidence":"Medium","gaps":["Single cell line; mechanism of MMP2 suppression undefined","No receptor identification","No in vivo validation"]},{"year":2015,"claim":"How SEMA3G transcription is controlled and which receptor mediates its endothelial effects was resolved by demonstrating that PPARγ directly binds the SEMA3G promoter and that SEMA3G promotes endothelial migration through NRP2, establishing the first transcriptional regulator and receptor for SEMA3G.","evidence":"ChIP for PPARγ at SEMA3G promoter, reporter assay, siRNA knockdown, anti-NRP2 neutralizing antibody in HUVEC migration assay","pmids":["25335934"],"confidence":"High","gaps":["Whether NRP2 acts alone or with a plexin co-receptor was not determined","Downstream signaling from NRP2 in endothelial cells not characterized"]},{"year":2020,"claim":"Two studies expanded SEMA3G biology beyond cancer and vasculature: one showed SEMA3G promotes adipogenesis via PI3K/Akt/GSK3β and AMPK/SREBP-1c pathways in adipose and liver tissue with in vivo metabolic consequences, while another identified a human loss-of-function SEMA3G mutation disrupting PlexinA binding and GnRH neuron migration, linking SEMA3G to neuroendocrine development.","evidence":"shRNA knockdown in 3T3-L1 preadipocytes and HFD mouse model (adipogenesis); exome sequencing, homology modeling, and GnRH neuron migration assay (neuroendocrine)","pmids":["31648186","32365351"],"confidence":"Medium","gaps":["Adipogenesis study from single lab without independent replication","GnRH study relies on structural modeling rather than direct binding measurement for PlexinA interaction","Whether SEMA3G signals through NRP or PlexinA in adipocytes is unknown"]},{"year":2023,"claim":"The paracrine signaling axis from endothelial-derived SEMA3G to vascular smooth muscle cells was mechanistically dissected, revealing that SEMA3G acts through NRP2/PlexinA1 to inhibit LATS1 and activate YAP, driving VSMC proliferation — the first defined intracellular pathway for SEMA3G-driven proliferation.","evidence":"Recombinant SEMA3G treatment of HASMCs with receptor-specific inhibition, verteporfin (YAP inhibitor) rescue, in vivo diabetic femoral injury model","pmids":["36720439"],"confidence":"High","gaps":["How NRP2/PlexinA1 engagement leads to LATS1 inhibition is not defined","Whether this pathway operates outside diabetic vascular injury is untested"]},{"year":2024,"claim":"The upstream transcriptional control of SEMA3G in pulmonary endothelium and its anti-angiogenic mechanism were defined: BMP9 signals through ALK1/Smad1/Smad5 and SOX17 to upregulate SEMA3G, which inhibits VEGFR2 phosphorylation, positioning SEMA3G as an effector of the BMP9 anti-angiogenic program.","evidence":"Sequential siRNA knockdowns of BMPR2, ACTR2A, Smad1, Smad5, SOX17, and SEMA3G with VEGFR2 phosphorylation and endothelial functional assays","pmids":["38795838"],"confidence":"High","gaps":["Direct SOX17 binding to SEMA3G promoter not confirmed by ChIP","Whether SEMA3G inhibits VEGFR2 through direct binding or indirect receptor crosstalk is unresolved"]},{"year":2025,"claim":"SEMA3G was identified as an immune checkpoint ligand: it binds NRP1 with higher affinity than NRP2 on CD8⁺ T cells, and its genetic deletion or antibody blockade restores T-cell cytotoxicity and suppresses tumor growth, establishing a direct immunosuppressive function.","evidence":"Receptor affinity screening, CRISPR/Cas9 knockout, neutralizing antibody blockade, CD8⁺ T-cell cytotoxicity assays, in vivo tumor models","pmids":["39652581"],"confidence":"High","gaps":["Downstream signaling in T cells upon NRP1 engagement not characterized","Contribution of NRP2 vs NRP1 in different immune cell subsets unclear"]},{"year":2025,"claim":"The deepest mechanistic dissection of SEMA3G signaling was achieved in glioblastoma: endothelial-derived SEMA3G activates NRP2/PLXNA1 on GSCs, inactivating Cdc42 and releasing WWP2 to ubiquitinate and degrade c-Myc, thereby suppressing cancer stemness — the first complete signaling cascade from SEMA3G receptor to a transcription factor substrate.","evidence":"Co-IP, genetic Sema3G deletion in ECs, ubiquitination assays, recombinant protein treatment, in vivo GBM survival model","pmids":["40533501"],"confidence":"High","gaps":["Whether Cdc42–WWP2 dissociation is a general mechanism downstream of all SEMA3G/PlexinA signaling or GSC-specific is unknown","Structural basis for SEMA3G-induced Cdc42 inactivation not defined"]},{"year":null,"claim":"Key unresolved questions include the structural basis for SEMA3G's differential receptor selectivity (NRP1 vs NRP2), how SEMA3G inhibits VEGFR2 phosphorylation (direct vs indirect), and whether the diverse downstream cascades (YAP, Cdc42/WWP2, PI3K/Akt) converge on shared proximal signaling events or represent entirely context-specific pathways.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure of SEMA3G–neuropilin complex","No unified model reconciling pro-migratory and anti-migratory effects across cell types","In vivo genetic models (conditional knockouts) in most tissue contexts are lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[1,4,6,7,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,7,8]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,1,4,6,7,8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4,6,7,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3]}],"complexes":[],"partners":["NRP1","NRP2","PLXNA1","WWP2","CDC42","SOX17"],"other_free_text":[]},"mechanistic_narrative":"SEMA3G is a secreted class-3 semaphorin that signals through neuropilin (NRP1, NRP2) and PlexinA receptors to regulate cell migration, proliferation, and differentiation across vascular, neural, metabolic, and immune contexts. In endothelial cells, BMP9/ALK1/Smad/SOX17 signaling transcriptionally induces SEMA3G, which then inhibits VEGFR2 phosphorylation to suppress VEGF-driven angiogenesis [PMID:38795838], while PPARγ directly activates the SEMA3G promoter to promote NRP2-dependent endothelial migration [PMID:25335934]. SEMA3G exerts paracrine effects on neighboring cells: it activates YAP via NRP2/PlexinA1–LATS1 inhibition to drive vascular smooth muscle cell proliferation [PMID:36720439], induces c-Myc ubiquitination through a NRP2/PLXNA1–Cdc42–WWP2 cascade to suppress glioblastoma stem cell stemness [PMID:40533501], binds NRP1 on CD8⁺ T cells to function as an immune checkpoint ligand suppressing cytotoxic immunity [PMID:39652581], and promotes adipogenesis via PI3K/Akt/GSK3β and AMPK/SREBP-1c signaling [PMID:31648186]. A loss-of-function SEMA3G mutation that disrupts PlexinA binding impairs GnRH neuron migration, linking SEMA3G deficiency to disorders of GnRH neuron development [PMID:32365351]."},"prefetch_data":{"uniprot":{"accession":"Q9NS98","full_name":"Semaphorin-3G","aliases":["Semaphorin sem2"],"length_aa":782,"mass_kda":86.7,"function":"Has chemorepulsive activities for sympathetic axons. Ligand of NRP2 (By similarity)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q9NS98/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SEMA3G","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SEMA3G","total_profiled":1310},"omim":[{"mim_id":"620997","title":"SEMAPHORIN 3G; SEMA3G","url":"https://www.omim.org/entry/620997"},{"mim_id":"147950","title":"HYPOGONADOTROPIC HYPOGONADISM 2 WITH OR WITHOUT ANOSMIA; HH2","url":"https://www.omim.org/entry/147950"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"adipose tissue","ntpm":67.3},{"tissue":"breast","ntpm":55.7}],"url":"https://www.proteinatlas.org/search/SEMA3G"},"hgnc":{"alias_symbol":["FLJ00014","sem2"],"prev_symbol":[]},"alphafold":{"accession":"Q9NS98","domains":[{"cath_id":"2.130.10.10","chopping":"53-422","consensus_level":"medium","plddt":92.8777,"start":53,"end":422},{"cath_id":"3.30.1680.10","chopping":"521-574","consensus_level":"medium","plddt":87.2635,"start":521,"end":574},{"cath_id":"2.60.40.10","chopping":"590-683","consensus_level":"high","plddt":89.6319,"start":590,"end":683}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NS98","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NS98-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NS98-F1-predicted_aligned_error_v6.png","plddt_mean":82.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SEMA3G","jax_strain_url":"https://www.jax.org/strain/search?query=SEMA3G"},"sequence":{"accession":"Q9NS98","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NS98.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NS98/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NS98"}},"corpus_meta":[{"pmid":"22562223","id":"PMC_22562223","title":"Effects of SEMA3G on migration and invasion of glioma cells.","date":"2012","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/22562223","citation_count":45,"is_preprint":false},{"pmid":"23317273","id":"PMC_23317273","title":"Quantitative real-time RT-PCR of ITGA7, SVEP1, TNS1, LPHN3, SEMA3G, KLB and MMP13 mRNA expression in breast cancer.","date":"2012","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/23317273","citation_count":24,"is_preprint":false},{"pmid":"31648186","id":"PMC_31648186","title":"Mechanism of SEMA3G knockdown-mediated attenuation of high-fat diet-induced obesity.","date":"2020","source":"The Journal of endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/31648186","citation_count":20,"is_preprint":false},{"pmid":"25335934","id":"PMC_25335934","title":"PPAR-γ promotes endothelial cell migration by inducing the expression of Sema3g.","date":"2015","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25335934","citation_count":17,"is_preprint":false},{"pmid":"32365351","id":"PMC_32365351","title":"A Novel SEMA3G Mutation in Two Siblings Affected by Syndromic GnRH Deficiency.","date":"2020","source":"Neuroendocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/32365351","citation_count":17,"is_preprint":false},{"pmid":"15939377","id":"PMC_15939377","title":"Developmental expression of sema3G, a novel zebrafish semaphorin.","date":"2005","source":"Gene expression patterns : GEP","url":"https://pubmed.ncbi.nlm.nih.gov/15939377","citation_count":14,"is_preprint":false},{"pmid":"21307099","id":"PMC_21307099","title":"The C. elegans SoxC protein SEM-2 opposes differentiation factors to promote a proliferative blast cell fate in the postembryonic mesoderm.","date":"2011","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/21307099","citation_count":13,"is_preprint":false},{"pmid":"32160477","id":"PMC_32160477","title":"Isolation of Bacillus subtilis strain SEM-2 from silkworm excrement and characterisation of its antagonistic effect against Fusarium spp.","date":"2020","source":"Canadian journal of microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32160477","citation_count":11,"is_preprint":false},{"pmid":"36720439","id":"PMC_36720439","title":"Sema3G activates YAP and promotes VSMCs proliferation and migration via Nrp2/PlexinA1.","date":"2023","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/36720439","citation_count":9,"is_preprint":false},{"pmid":"36882799","id":"PMC_36882799","title":"MiR-146b-5p/SEMA3G regulates epithelial-mesenchymal transition in clear cell renal cell carcinoma.","date":"2023","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/36882799","citation_count":7,"is_preprint":false},{"pmid":"39652581","id":"PMC_39652581","title":"SEMA3G-NRP1 Signaling Functions as an Immune Checkpoint That Enables Tumor Immune Evasion by Impairing T-cell Cytotoxicity.","date":"2025","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/39652581","citation_count":7,"is_preprint":false},{"pmid":"28614700","id":"PMC_28614700","title":"The C. elegans Spalt-like protein SEM-4 functions through the SoxC transcription factor SEM-2 to promote a proliferative blast cell fate in the postembryonic mesoderm.","date":"2017","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/28614700","citation_count":7,"is_preprint":false},{"pmid":"38795838","id":"PMC_38795838","title":"SEMA3G regulates BMP9 inhibition of VEGF-mediated migration and network formation in pulmonary endothelial cells.","date":"2024","source":"Vascular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38795838","citation_count":6,"is_preprint":false},{"pmid":"38192809","id":"PMC_38192809","title":"Mechanism of action of Shaoyao-Gancao decoction in relieving chronic inflammatory pain via Sema3G protein regulation in the dorsal root ganglion.","date":"2023","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/38192809","citation_count":4,"is_preprint":false},{"pmid":"40533501","id":"PMC_40533501","title":"Endothelial cells-derived SEMA3G suppresses glioblastoma stem cells by inducing c-Myc degradation.","date":"2025","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/40533501","citation_count":2,"is_preprint":false},{"pmid":"41312929","id":"PMC_41312929","title":"The Expression of Semaphorin 3G (SEMA3G) Protein in Breast Cancer Tissue by the Immunohistochemistry Method.","date":"2025","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/41312929","citation_count":0,"is_preprint":false},{"pmid":"41270983","id":"PMC_41270983","title":"BMSC-EVs improve post-stroke cognition by promoting regionally distinct synaptic repair via Sema3G-Nrp2/PlexinA4 Signaling.","date":"2025","source":"Experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/41270983","citation_count":0,"is_preprint":false},{"pmid":"41504904","id":"PMC_41504904","title":"Exploring Exercise-Linked Neurovascular Unit Adaptations and Sema3G Pathway Involvement in Rats with Vascular Cognitive Impairment.","date":"2026","source":"Neurochemical research","url":"https://pubmed.ncbi.nlm.nih.gov/41504904","citation_count":0,"is_preprint":false},{"pmid":"38075209","id":"PMC_38075209","title":"SEM2: Introducing mechanics in cell and tissue modeling using coarse-grained homogeneous particle dynamics.","date":"2023","source":"APL bioengineering","url":"https://pubmed.ncbi.nlm.nih.gov/38075209","citation_count":0,"is_preprint":false},{"pmid":"39612117","id":"PMC_39612117","title":"1H, 13C, and 15N resonance assignments of the amyloidogenic peptide SEM2(49-107) by NMR spectroscopy.","date":"2024","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/39612117","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10964,"output_tokens":2334,"usd":0.033951},"stage2":{"model":"claude-opus-4-6","input_tokens":5654,"output_tokens":2592,"usd":0.139605},"total_usd":0.173556,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"SEMA3G overexpression in U251MG glioma cells inhibits cell migration and invasion, and reduces MMP2 activity, functioning through both cell-autonomous and paracrine mechanisms.\",\n      \"method\": \"Stable overexpression in glioma cells, conditioned media treatment, Matrigel invasion assay, MMP2 activity assay\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — clean overexpression/KD with defined cellular phenotype and mechanistic readout (MMP2 activity), single lab\",\n      \"pmids\": [\"22562223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PPAR-γ directly binds the SEMA3G promoter and transcriptionally activates SEMA3G expression, which in turn promotes endothelial cell migration via neuropilin-2 (NRP2) as the receptor.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), transient transfection reporter assay, siRNA knockdown, anti-NRP2 neutralizing antibody, HUVEC migration assay\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal ChIP + reporter assay + receptor blockade with functional rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"25335934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SEMA3G promotes adipocyte differentiation and lipogenesis; its knockdown inhibits high-fat diet-induced obesity. SEMA3G acts through PI3K/Akt/GSK3β signaling in adipose tissue and the AMPK/SREBP-1c pathway in liver.\",\n      \"method\": \"shRNA lentiviral knockdown in 3T3-L1 cells and primary preadipocytes, in vivo HFD mouse model, pathway inhibitor assays\",\n      \"journal\": \"The Journal of endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo KD with defined metabolic phenotype and pathway identification, single lab\",\n      \"pmids\": [\"31648186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A loss-of-function SEMA3G mutation alters binding to PlexinA receptors and attenuates migration of immortalized GnRH neurons; SEMA3G is expressed along the migratory route of GnRH neurons and in the developing pituitary, regulating GnRH neuron development.\",\n      \"method\": \"Homozygosity mapping, exome sequencing, homology modelling, cell migration assay in immortalized GnRH neurons, in vivo mouse expression analysis\",\n      \"journal\": \"Neuroendocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in silico structural modeling combined with in vitro migration assay and in vivo expression, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"32365351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ECs-derived SEMA3G promotes vascular smooth muscle cell (VSMC) proliferation and migration via Nrp2/PlexinA1 receptors, mechanistically by inhibiting LATS1 and activating YAP, leading to upregulation of cyclin D1, cyclin E, MMP2, and MMP9.\",\n      \"method\": \"Recombinant SEMA3G treatment of HASMCs, receptor inhibition (anti-Nrp2/PlexinA1), YAP inhibitor (verteporfin), western blot, cell cycle analysis, in vivo diabetic mouse femoral injury model\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — receptor-specific inhibition, pathway inhibitor rescue, in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"36720439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"miR-146b-5p directly targets and suppresses SEMA3G expression in clear cell renal cell carcinoma; SEMA3G loss promotes EMT, cell migration, and invasion via Notch and TGF-β signaling pathways.\",\n      \"method\": \"Dual-luciferase reporter assay, qRT-PCR, western blot (EMT markers, Notch/TGF-β pathway proteins, MMP2/9), Transwell assay, wound healing assay\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct miRNA-target validation by dual-luciferase plus functional assays, single lab\",\n      \"pmids\": [\"36882799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BMP9 transcriptionally upregulates SEMA3G via ALK1/Smad1/Smad5 canonical signaling and the transcription factor SOX17 in pulmonary endothelial cells; SEMA3G then inhibits VEGFR2 phosphorylation and VEGF-mediated endothelial migration and network formation.\",\n      \"method\": \"Knockdown studies (siRNA for BMPR2, ACTR2A, Smad1, Smad5, SOX17, SEMA3G), VEGFR2 phosphorylation assay, functional endothelial migration and network formation assays, recombinant SEMA3G treatment\",\n      \"journal\": \"Vascular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic knockdowns defining pathway hierarchy plus receptor phosphorylation assay and functional rescue with recombinant protein, multiple orthogonal methods\",\n      \"pmids\": [\"38795838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SEMA3G acts as an immune checkpoint ligand binding NRP1 (with stronger affinity than NRP2) to suppress CD8+ T-cell cytotoxicity; CRISPR/Cas9 deletion or neutralizing antibody blockade of SEMA3G restores T-cell cytotoxicity and inhibits tumor growth in vivo.\",\n      \"method\": \"Receptor binding/affinity screening, CRISPR/Cas9 knockout, neutralizing antibody blockade, CD8+ T-cell cytotoxicity assay, in vivo tumor growth assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — receptor affinity determination combined with genetic KO and antibody blockade with in vitro and in vivo functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"39652581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ECs-derived SEMA3G activates NRP2/PLXNA1 on glioblastoma stem cells (GSCs) in a paracrine manner, inducing Cdc42 inactivation and dissociation of Cdc42 from WWP2; free WWP2 then ubiquitinates c-Myc, leading to c-Myc degradation and impairment of GSC stemness.\",\n      \"method\": \"Co-IP, genetic deletion of Sema3G in ECs, overexpression, recombinant SEMA3G protein treatment, ubiquitination assay, in vivo GBM mouse survival model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical mechanism (ubiquitination assay, Co-IP dissociation of Cdc42/WWP2) combined with genetic KO and in vivo survival, rigorous mechanistic dissection\",\n      \"pmids\": [\"40533501\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SEMA3G is a secreted class-3 semaphorin that signals through NRP1, NRP2, and PlexinA family receptors to regulate diverse cellular processes: it inhibits VEGFR2 phosphorylation downstream of BMP9/ALK1/Smad/SOX17 signaling to suppress angiogenesis; activates YAP via NRP2/PlexinA1 to promote vascular smooth muscle cell proliferation; induces c-Myc ubiquitination via a NRP2/PLXNA1–Cdc42–WWP2 cascade to suppress glioblastoma stem cell stemness; binds NRP1 on T cells to suppress cytotoxic immunity; regulates GnRH neuron migration through PlexinA receptors; and promotes adipogenesis via PI3K/Akt/GSK3β and AMPK/SREBP-1c pathways.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SEMA3G is a secreted class-3 semaphorin that signals through neuropilin (NRP1, NRP2) and PlexinA receptors to regulate cell migration, proliferation, and differentiation across vascular, neural, metabolic, and immune contexts. In endothelial cells, BMP9/ALK1/Smad/SOX17 signaling transcriptionally induces SEMA3G, which then inhibits VEGFR2 phosphorylation to suppress VEGF-driven angiogenesis [PMID:38795838], while PPARγ directly activates the SEMA3G promoter to promote NRP2-dependent endothelial migration [PMID:25335934]. SEMA3G exerts paracrine effects on neighboring cells: it activates YAP via NRP2/PlexinA1–LATS1 inhibition to drive vascular smooth muscle cell proliferation [PMID:36720439], induces c-Myc ubiquitination through a NRP2/PLXNA1–Cdc42–WWP2 cascade to suppress glioblastoma stem cell stemness [PMID:40533501], binds NRP1 on CD8⁺ T cells to function as an immune checkpoint ligand suppressing cytotoxic immunity [PMID:39652581], and promotes adipogenesis via PI3K/Akt/GSK3β and AMPK/SREBP-1c signaling [PMID:31648186]. A loss-of-function SEMA3G mutation that disrupts PlexinA binding impairs GnRH neuron migration, linking SEMA3G deficiency to disorders of GnRH neuron development [PMID:32365351].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"The initial question of whether SEMA3G has functional relevance in tumor cell behavior was addressed by showing that overexpression in glioma cells inhibits migration and invasion through both cell-autonomous and paracrine mechanisms involving MMP2 suppression, establishing SEMA3G as a negative regulator of tumor cell motility.\",\n      \"evidence\": \"Stable overexpression in U251MG glioma cells with conditioned media transfer and Matrigel invasion assays\",\n      \"pmids\": [\"22562223\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cell line; mechanism of MMP2 suppression undefined\", \"No receptor identification\", \"No in vivo validation\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"How SEMA3G transcription is controlled and which receptor mediates its endothelial effects was resolved by demonstrating that PPARγ directly binds the SEMA3G promoter and that SEMA3G promotes endothelial migration through NRP2, establishing the first transcriptional regulator and receptor for SEMA3G.\",\n      \"evidence\": \"ChIP for PPARγ at SEMA3G promoter, reporter assay, siRNA knockdown, anti-NRP2 neutralizing antibody in HUVEC migration assay\",\n      \"pmids\": [\"25335934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NRP2 acts alone or with a plexin co-receptor was not determined\", \"Downstream signaling from NRP2 in endothelial cells not characterized\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Two studies expanded SEMA3G biology beyond cancer and vasculature: one showed SEMA3G promotes adipogenesis via PI3K/Akt/GSK3β and AMPK/SREBP-1c pathways in adipose and liver tissue with in vivo metabolic consequences, while another identified a human loss-of-function SEMA3G mutation disrupting PlexinA binding and GnRH neuron migration, linking SEMA3G to neuroendocrine development.\",\n      \"evidence\": \"shRNA knockdown in 3T3-L1 preadipocytes and HFD mouse model (adipogenesis); exome sequencing, homology modeling, and GnRH neuron migration assay (neuroendocrine)\",\n      \"pmids\": [\"31648186\", \"32365351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Adipogenesis study from single lab without independent replication\", \"GnRH study relies on structural modeling rather than direct binding measurement for PlexinA interaction\", \"Whether SEMA3G signals through NRP or PlexinA in adipocytes is unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The paracrine signaling axis from endothelial-derived SEMA3G to vascular smooth muscle cells was mechanistically dissected, revealing that SEMA3G acts through NRP2/PlexinA1 to inhibit LATS1 and activate YAP, driving VSMC proliferation — the first defined intracellular pathway for SEMA3G-driven proliferation.\",\n      \"evidence\": \"Recombinant SEMA3G treatment of HASMCs with receptor-specific inhibition, verteporfin (YAP inhibitor) rescue, in vivo diabetic femoral injury model\",\n      \"pmids\": [\"36720439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How NRP2/PlexinA1 engagement leads to LATS1 inhibition is not defined\", \"Whether this pathway operates outside diabetic vascular injury is untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The upstream transcriptional control of SEMA3G in pulmonary endothelium and its anti-angiogenic mechanism were defined: BMP9 signals through ALK1/Smad1/Smad5 and SOX17 to upregulate SEMA3G, which inhibits VEGFR2 phosphorylation, positioning SEMA3G as an effector of the BMP9 anti-angiogenic program.\",\n      \"evidence\": \"Sequential siRNA knockdowns of BMPR2, ACTR2A, Smad1, Smad5, SOX17, and SEMA3G with VEGFR2 phosphorylation and endothelial functional assays\",\n      \"pmids\": [\"38795838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct SOX17 binding to SEMA3G promoter not confirmed by ChIP\", \"Whether SEMA3G inhibits VEGFR2 through direct binding or indirect receptor crosstalk is unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"SEMA3G was identified as an immune checkpoint ligand: it binds NRP1 with higher affinity than NRP2 on CD8⁺ T cells, and its genetic deletion or antibody blockade restores T-cell cytotoxicity and suppresses tumor growth, establishing a direct immunosuppressive function.\",\n      \"evidence\": \"Receptor affinity screening, CRISPR/Cas9 knockout, neutralizing antibody blockade, CD8⁺ T-cell cytotoxicity assays, in vivo tumor models\",\n      \"pmids\": [\"39652581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling in T cells upon NRP1 engagement not characterized\", \"Contribution of NRP2 vs NRP1 in different immune cell subsets unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The deepest mechanistic dissection of SEMA3G signaling was achieved in glioblastoma: endothelial-derived SEMA3G activates NRP2/PLXNA1 on GSCs, inactivating Cdc42 and releasing WWP2 to ubiquitinate and degrade c-Myc, thereby suppressing cancer stemness — the first complete signaling cascade from SEMA3G receptor to a transcription factor substrate.\",\n      \"evidence\": \"Co-IP, genetic Sema3G deletion in ECs, ubiquitination assays, recombinant protein treatment, in vivo GBM survival model\",\n      \"pmids\": [\"40533501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Cdc42–WWP2 dissociation is a general mechanism downstream of all SEMA3G/PlexinA signaling or GSC-specific is unknown\", \"Structural basis for SEMA3G-induced Cdc42 inactivation not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for SEMA3G's differential receptor selectivity (NRP1 vs NRP2), how SEMA3G inhibits VEGFR2 phosphorylation (direct vs indirect), and whether the diverse downstream cascades (YAP, Cdc42/WWP2, PI3K/Akt) converge on shared proximal signaling events or represent entirely context-specific pathways.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure of SEMA3G–neuropilin complex\", \"No unified model reconciling pro-migratory and anti-migratory effects across cell types\", \"In vivo genetic models (conditional knockouts) in most tissue contexts are lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [1, 4, 6, 7, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 4, 6, 7, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 6, 7, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NRP1\", \"NRP2\", \"PLXNA1\", \"WWP2\", \"CDC42\", \"SOX17\"],\n    \"other_free_text\": []\n  }\n}\n```"}