{"gene":"ACSL3","run_date":"2026-06-09T22:02:39","timeline":{"discoveries":[{"year":2010,"finding":"ACSL3 associates with the C-lobe of the Lyn kinase domain on the Golgi in a conformation-dependent manner (open conformation of Lyn required) and mediates Golgi export of Lyn to the plasma membrane; an ACSL3 mutant lacking the LR2 catalytic domain retains both Lyn-binding and Golgi export activity, indicating the function is independent of ACSL3 enzymatic activity.","method":"Co-immunoprecipitation, overexpression and siRNA knockdown of ACSL3 with live-cell trafficking assays, domain-deletion mutagenesis, confocal imaging","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays, functional domain mutagenesis, overexpression and knockdown with defined trafficking phenotype, single lab but multiple orthogonal methods","pmids":["20605918"],"is_preprint":false},{"year":2007,"finding":"Oncostatin M activates transcription of ACSL3 (and ACSL5) in hepatocytes through the ERK signaling pathway; increased ACSL3 activity partitions fatty acids into β-oxidation rather than triglyceride synthesis, reducing TG accumulation; siRNA knockdown of ACSL3/ACSL5 abrogates OM-enhanced fatty acid oxidation.","method":"Transcriptional reporter assays, siRNA knockdown, fatty acid oxidation assays in HepG2 cells and in vivo hamster model, acyl-CoA synthetase activity measurement","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA rescue, enzymatic activity assay, in vivo validation, multiple orthogonal methods in single lab","pmids":["17761945"],"is_preprint":false},{"year":2010,"finding":"LXR activation directly regulates ACSL3 transcription through a conserved LXR response element in the ACSL3 promoter in human placental trophoblast cells, increasing acyl-CoA synthetase activity and fatty acid uptake; ACSL3 silencing attenuates LXR-mediated increases in acyl-CoA synthetase activity.","method":"Promoter reporter assays with LXR response element identification, siRNA knockdown, acyl-CoA synthetase activity assay, fatty acid uptake assay","journal":"Journal of lipid research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — promoter mutagenesis/reporter assay combined with siRNA knockdown and enzymatic activity measurement, single lab, multiple orthogonal methods","pmids":["20219900"],"is_preprint":false},{"year":2011,"finding":"ACSL3 expression is induced by ER stress (tunicamycin) in hepatocytes; ACSL3 shRNA (but not ACSL1 shRNA) blocks ER stress-induced lipid accumulation; GSK-3β acts upstream of ACSL3 in this pathway, as GSK-3β inhibitors or shRNA suppress ACSL3 upregulation and lipid accumulation.","method":"shRNA knockdown, GSK-3β inhibitor treatment, lipid accumulation assays (Oil Red O), western blot in HuH-7 and HepG2 cells, hepatitis B virus mutant large surface protein model","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic (shRNA) and pharmacological dissection of pathway order, single lab, two orthogonal approaches","pmids":["21328461"],"is_preprint":false},{"year":2013,"finding":"ACSL3 mediates palmitic acid (PA)-induced osteoblastic differentiation and calcium deposition in vascular smooth muscle cells; ACS inhibitor or ACSL3 siRNA prevents PA-induced BMP-2 and Msx2 expression and calcium deposition; adenovirus-mediated ACSL3 overexpression enhances these effects; EPA inhibits ACSL3 expression and downstream osteoblastic gene induction.","method":"siRNA knockdown, adenovirus-mediated overexpression, ACS pharmacological inhibitor, qPCR for osteoblastic markers, calcium deposition assay, immunohistochemistry of human plaques","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic (siRNA) and gain-of-function (adenovirus overexpression) plus pharmacological inhibition with defined molecular phenotype, multiple orthogonal methods, single lab","pmids":["23840832"],"is_preprint":false},{"year":2017,"finding":"ACSL3 overexpression in androgen-dependent LNCaP prostate cancer cells upregulates AKR1C3 (involved in steroidogenesis converting DHEAS to testosterone) and downregulates the androgen-inactivating enzyme UGT2B, promoting intratumoral androgen synthesis and cell proliferation in response to DHEAS.","method":"ACSL3 overexpression in LNCaP cells, gene expression profiling, testosterone measurement by mass spectrometry, cell proliferation assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — overexpression with defined downstream gene expression and metabolite measurement, single lab, single main approach","pmids":["28771887"],"is_preprint":false},{"year":2017,"finding":"ACSL3 and ACSL4 are concentrated in insulin secretory granules of pancreatic beta cells; shRNA-mediated knockdown of ACSL3 or ACSL4 inhibits glucose-stimulated insulin secretion ~50% in INS-1 832/13 cells and in human pancreatic islets; ACSL3 knockdown preferentially reduces arachidonate over palmitate as substrate.","method":"Subcellular fractionation, immunostaining, stable shRNA knockdown cell lines, glucose-stimulated insulin secretion assay, ACSL enzyme activity assay, phospholipid profiling","journal":"Archives of biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation, stable knockdown with functional assay validated in human islets, enzymatic activity confirmation, multiple orthogonal methods, single lab","pmids":["28193492"],"is_preprint":false},{"year":2018,"finding":"Upper small intestinal ACSL3 expression is required for fatty acid-dependent pre-absorptive signaling that regulates glucose homeostasis; high-fat feeding reduces ACSL3 expression and impairs fatty acid sensing; restoration of Lactobacillus gasseri increases ACSL3 expression and restores fatty acid sensing and glucose tolerance in rodents.","method":"Upper small intestinal infusion of lipids, surgical duodenal cannulation, in vivo glucose clamps, ACSL3 knockdown/expression measurements, microbiota transplantation, L. gasseri probiotic administration","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo glucose clamp measurements with genetic pathway dissection, microbiota manipulation with defined molecular readout, multiple orthogonal approaches, single lab","pmids":["29514066"],"is_preprint":false},{"year":2018,"finding":"Endogenous ACSL3 in fibrosarcoma and breast cancer cells localizes to the trans-Golgi network/endosomal compartments, distinct from ACSL4 which follows the endoplasmic reticulum pattern; both isoforms associate with lipid droplets.","method":"Subcellular fractionation, confocal immunofluorescence imaging, immunohistochemistry of tumor arrays","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation combined with confocal imaging, replicated across two cell lines, single lab","pmids":["29450800"],"is_preprint":false},{"year":2020,"finding":"ACSL3 channels arachidonic acid (AA) into phosphatidylinositols, providing a substrate pool for LPIAT1 to sustain elevated prostaglandin synthesis in non-small cell lung cancer; LPIAT1 knockdown suppresses proliferation and in vivo tumorigenesis, defining an ACSL3-LPIAT1 axis for prostaglandin production.","method":"ACSL3 and LPIAT1 knockdown in lung cancer cell lines and KrasG12D mouse models, lipidomics, prostaglandin measurement, proliferation and anchorage-independent growth assays, in vivo tumorigenesis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockdown validated in cell lines and clinically relevant in vivo mouse model with lipidomic substrate tracing, multiple orthogonal methods, replicated across model systems","pmids":["32034305"],"is_preprint":false},{"year":2020,"finding":"ACSL3 is required for lipid droplet accumulation from exogenous serum-derived fatty acids (not de novo lipogenesis) in clear cell renal cell carcinoma cells; genetic or pharmacologic ACSL3 suppression is cytotoxic to ccRCC in vitro and reduces tumor weight in an orthotopic mouse model; ACSL3 inhibition decreases ferroptosis susceptibility in a manner dependent on exogenous fatty acid composition.","method":"siRNA/shRNA knockdown, pharmacological inhibition, isotope-tracing lipidomics, Oil Red O staining, cell viability assays, orthotopic mouse tumor model, FACS-based ferroptosis assays","journal":"Cancer & metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological inhibition with isotope tracing, in vitro and in vivo validation, multiple orthogonal methods, single lab","pmids":["36192773"],"is_preprint":false},{"year":2020,"finding":"ACSL3 is a direct binding partner of GABARAPL2 (via LC3-interacting regions); through this interaction GABARAPL2 is recruited to the ER, anchoring UBA5 (UFM1-activating enzyme) at the ER; ACSL3 depletion and lipid droplet induction affect abundance of ufmylation components and ER-phagy, establishing ACSL3 as a regulator of the UFM1 conjugation pathway.","method":"CRISPR/Cas9 endogenous tagging of ATG8 proteins, interaction proteomics (affinity purification–mass spectrometry), co-immunoprecipitation, knockdown experiments","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — endogenous protein tagging by CRISPR, affinity purification–MS interactome with Co-IP validation, functional knockdown, multiple orthogonal methods, single lab","pmids":["32843575"],"is_preprint":false},{"year":2020,"finding":"ACSL3 knockout in pancreatic ductal adenocarcinoma hinders tumor progression, reduces tumor fibrosis, reduces immunosuppressive cell infiltration, and increases cytotoxic T cell infiltration; this is mediated at least in part through decreased PAI-1 secretion from tumor cells, defining an ACSL3–PAI-1 signaling axis.","method":"Acsl3 genetic knockout in mouse PDAC models, in vivo tumor growth assays, flow cytometry of tumor-infiltrating immune cells, PAI-1 measurement, PAI-1 pharmacological inhibition with chemo/immunotherapy response assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO in vivo with multiple defined phenotypic readouts (fibrosis, immunity, PAI-1 secretion), pharmacological rescue experiment, single lab but rigorous in vivo model","pmids":["33127675"],"is_preprint":false},{"year":2021,"finding":"Rab18 interacts with ACSL3 on lipid droplets and promotes ACSL3 LD localization; Rab18 also binds PLIN2, which recruits Rab18 from ER to LDs; the Rab18-PLIN2-ACSL3 complex regulates triacylglycerol levels and lipid droplet dynamics in myoblast cells.","method":"Co-immunoprecipitation, Rab18 overexpression and knockdown, lipid droplet staining, TAG quantification, confocal imaging in C2C12 cells","journal":"Biochimica et biophysica acta. Molecular and cell biology of lipids","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP binding shown, overexpression and loss-of-function with defined phenotype, single lab, single main binding method","pmids":["33713834"],"is_preprint":false},{"year":2022,"finding":"MAT2A mediates ferroptosis resistance in gastric cancer by producing S-adenosylmethionine (SAM), which upregulates ACSL3 expression via H3K4me3 trimethylation at the ACSL3 promoter, thereby increasing resistance to ferroptosis.","method":"Pharmacological and genetic blockade of methionine cycle, chromatin immunoprecipitation (H3K4me3 at ACSL3 promoter), gene expression analysis, in vitro and in vivo ferroptosis assays","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for H3K4me3 at ACSL3 promoter combined with genetic/pharmacologic methionine cycle blockade and ferroptosis phenotype, single lab, two orthogonal methods","pmids":["35182729"],"is_preprint":false},{"year":2023,"finding":"BRD4 controls the splicing efficiency of ACSL3 pre-mRNA by recruiting SRPK2 to assemble a splicing catalytic platform; the AMP-binding domain of ACSL3 influences arachidonic acid synthesis and thus determines susceptibility to erastin-induced ferroptosis in osteosarcoma cells.","method":"BRD4 inhibition (in vitro and in vivo), SRPK2 co-IP, RT-PCR splicing analysis, ACSL3 domain mutants, arachidonic acid measurement, ferroptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — splicing analysis combined with domain mutagenesis and functional ferroptosis assay, single lab, multiple orthogonal approaches","pmids":["37993451"],"is_preprint":false},{"year":2023,"finding":"FTO demethylates m6A modifications on ACSL3 mRNA (and GPX4 mRNA), decreasing their stability and expression, thereby sensitizing oral squamous cell carcinoma cells to ferroptosis in vitro and in vivo.","method":"FTO overexpression/knockdown, m6A methylation analysis (MeRIP), mRNA stability assays, ferroptosis assays (lipid ROS, cell viability) in vitro and in vivo","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP for m6A site identification combined with mRNA stability assays and functional ferroptosis readout in vitro and in vivo, single lab","pmids":["38003537"],"is_preprint":false},{"year":2024,"finding":"ANKRD1 directly binds ACSL3 and promotes its degradation via K63-linked ubiquitination catalyzed by the E3 ligase TRIM25, reducing ACSL3 protein levels, amplifying lipid peroxidation and ferroptosis, and exacerbating renal ischemia-reperfusion injury.","method":"Immunoprecipitation-mass spectrometry to identify ANKRD1 interactors, Co-IP and proximity ligation assay for ANKRD1-ACSL3 and TRIM25-ACSL3 interactions, ubiquitination assays (K63-linkage), ANKRD1 knockdown (rAAV9) in vivo, siRNA in vitro, cell viability and lipid peroxidation assays","journal":"Clinical and translational medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — IP-MS interactome, reciprocal Co-IP, ubiquitination assay with linkage specificity, in vivo genetic KD with defined phenotype, multiple orthogonal methods, single lab","pmids":["39285846"],"is_preprint":false},{"year":2024,"finding":"MEF2D directly binds the promoter region of ACSL3 and transcriptionally upregulates ACSL3 expression, inhibiting ferroptosis and enhancing sorafenib resistance in hepatocellular carcinoma.","method":"Promoter binding assays (ChIP or EMSA), gene expression analysis, ACSL3 silencing in sorafenib-resistant HCC cells, ferroptosis level assessment","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — promoter binding assay plus functional knockdown, single lab, single binding approach","pmids":["39744125"],"is_preprint":false},{"year":2024,"finding":"METTL3 in cancer-associated fibroblast-derived exosomes induces m6A modification on ACSL3 mRNA, stabilizing ACSL3 expression, which promotes colorectal cancer cell proliferation, metastasis, and suppresses ferroptosis; METTL3 knockdown in CAFs reverses these effects and is rescued by ACSL3 overexpression.","method":"Methylated RNA immunoprecipitation (MeRIP), dual-luciferase reporter assay, exosome isolation, METTL3 knockdown in CAFs, ACSL3 overexpression rescue, in vitro and in vivo tumor models","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP confirms m6A site, rescue experiment validates mechanistic hierarchy, in vivo confirmation, single lab","pmids":["39160584"],"is_preprint":false},{"year":2024,"finding":"ACSL3 interacts with YES1 (Src-family kinase) and suppresses its activation (phospho-Tyr419), consequently inhibiting YAP1 nuclear colocalization and transcriptional complex formation in breast cancer cells; ACSL3 knockdown promotes cell proliferation, migration, and EMT.","method":"Co-immunoprecipitation for ACSL3-YES1 interaction, phospho-YES1 measurement, ACSL3 knockdown/overexpression, YAP1 nuclear localization assay, in vitro and in vivo functional assays","journal":"Cancer biology & medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP for ACSL3-YES1 binding plus phosphorylation measurement and defined downstream signaling phenotype, single lab","pmids":["38953696"],"is_preprint":false},{"year":2025,"finding":"TNFAIP3 promotes ACSL3 degradation via NEDD4-mediated ubiquitination, reducing ACSL3 levels, enhancing lipid peroxidation and ferroptosis in neurons after traumatic brain injury; TNFAIP3 overexpression increases neuronal cell death, while TNFAIP3 knockdown (AAV-shTNFAIP3) alleviates ferroptosis and cognitive impairment.","method":"Co-IP for TNFAIP3-ACSL3 interaction, ubiquitination assay with NEDD4, TNFAIP3 overexpression/knockdown, AAV-shTNFAIP3 in mouse TBI model, lipid peroxidation and ferroptosis markers","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP combined with ubiquitination assay identifying the E3 ligase and in vivo rescue, single lab","pmids":["39743027"],"is_preprint":false},{"year":2025,"finding":"HRD1 (an ER-associated E3 ubiquitin ligase) ubiquitinates ACSL3 and promotes its proteasomal degradation; HRD1 knockdown increases ACSL3 levels, suppresses fatty acid synthesis, promotes fatty acid oxidation, and alleviates alcohol-induced hepatic injury and steatosis.","method":"AAV9-shRNA knockdown of HRD1 and ACSL3 in mice, siRNA in HepG2 cells, co-immunoprecipitation for HRD1-ACSL3 interaction, ubiquitination assay, lipid metabolism measurements, Lieber-DeCarli ethanol diet model","journal":"Toxicology letters","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP combined with ubiquitination assay plus in vivo genetic knockdown with rescue phenotype, multiple orthogonal methods, single lab","pmids":["41130543"],"is_preprint":false},{"year":2025,"finding":"SUMO2 directly binds ACSL3 and inhibits its entry into the ubiquitin-proteasome degradation pathway, stabilizing ACSL3 protein and thereby suppressing ferroptosis in hepatocellular carcinoma cells; ACSL3 knockdown in SUMO2-overexpressing cells reverses SUMO2's anti-ferroptotic effect.","method":"Co-immunoprecipitation for SUMO2-ACSL3 interaction, ACSL3 ubiquitination assay, SUMO2 overexpression/knockdown, ACSL3 rescue knockdown, ferroptosis marker assays","journal":"Discover oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP for binding, ubiquitination assay for mechanism, rescue experiment for pathway order, single lab","pmids":["40526170"],"is_preprint":false},{"year":2025,"finding":"ACSL3 knockdown impairs starvation-induced autophagy and causes formation of enlarged autophagosome-like structures negative for WIPI2; ACSL3 overexpression induces WIPI2-positive but LC3-negative dots under normal nutrition; both effects are independent of ACSL3 enzymatic activity, suggesting ACSL3 functions in formation of fusion-competent autophagosomal membranes at a stage distinct from ACSL4.","method":"Knockdown and overexpression of ACSL3 and ACSL4, autophagy induction by starvation, immunofluorescence for WIPI2, LC3, FIP200, LC3 lipidation assay, enzymatic activity-dead mutants","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockdown and gain-of-function with domain/activity mutants and multiple autophagy marker readouts, single lab, multiple orthogonal methods","pmids":["40728409"],"is_preprint":false},{"year":2025,"finding":"ACSL3 is required for lipid droplet biogenesis during starvation and for formation of functional autophagosomes; under starvation ACSL3 is regulated by SYNTAXIN17; ACSL3 functions at an early autophagy stage (formation of autophagosomes) independently of its enzymatic activity.","method":"Knockdown of ACSL3, immunofluorescence for autophagy markers (FIP200, WIPI2, LC3), lipid droplet staining, genetic epistasis with SYNTAXIN17","journal":"Autophagy reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — knockdown with multiple marker readouts, single lab, overlap with companion paper (PMID 40728409)","pmids":["41346954"],"is_preprint":false},{"year":2025,"finding":"Vitamin A and ATRA directly target ACSL3 and enhance its enzymatic activity; this ACSL3-dependent mechanism increases the MUFA/PUFA ratio in phospholipids, preventing lipid peroxidation and suppressing ferroptosis; vitamin A and its analogue D3 extend C. elegans lifespan in an ACSL3-dependent manner.","method":"Biochemical binding and enzymatic activity assays, phospholipid lipidomics (MUFA/PUFA ratio), ferroptosis assays, VA analogue structure-activity relationship, C. elegans lifespan assay with ACSL3 genetic dependence","journal":"Acta pharmaceutica Sinica. B","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic activity assay with drug binding, lipidomics confirming substrate mechanism, in vivo genetic dependence assay, multiple orthogonal methods, single lab","pmids":["41909752"],"is_preprint":false},{"year":2025,"finding":"ACSL3 promotes synthesis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which activates the PPARα pathway and enhances transcription of downstream lipid metabolism genes, promoting hepatocellular carcinoma growth and metastasis by accelerating lipid catabolism and anabolism.","method":"Proteomic and scRNA-seq analysis, ACSL3 siRNA/nanoparticle-mediated knockdown, lipidomics (POPC measurement), PPARα pathway reporter assays, in vitro and in vivo tumor models","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lipidomics identifies specific lipid product combined with pathway reporter assay and in vivo genetic knockdown, single lab","pmids":["40059153"],"is_preprint":false},{"year":2025,"finding":"HNRNPC binds to ACSL3 RNA and promotes exon 10 skipping (alternative splicing), generating a short ACSL3-S isoform; m6A modification at the ACSL3 mRNA enhances HNRNPC binding; FBXW11 acts as an E3 ubiquitin ligase to ubiquitinate and degrade HNRNPC; HNRNPC knockdown in mice alleviates preeclampsia symptoms and dysregulates ferroptosis markers.","method":"RT-PCR and RT-qPCR for splicing analysis, co-IP for HNRNPC-ACSL3 RNA binding, m6A site mutation, in vitro ubiquitination assay, FBXW11 co-IP, mouse preeclampsia model with HNRNPC knockdown","journal":"Journal of hypertension","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — splicing assay with m6A mutation to confirm binding, in vitro ubiquitination, in vivo phenotype, multiple methods, single lab","pmids":["41037014"],"is_preprint":false},{"year":2025,"finding":"METTL7B promotes m6A modification on ACSL3 mRNA, stabilizing its expression and inhibiting erastin-induced ferroptosis in bladder cancer cells; METTL7B knockdown reduces ACSL3 protein levels and induces ferroptosis; ACSL3 overexpression rescues the pro-ferroptotic effect of METTL7B knockdown.","method":"MeRIP for m6A modification, METTL7B knockdown/overexpression, ACSL3 expression analysis, ferroptosis assays (lipid ROS, Fe2+, MDA), ACSL3 rescue experiment, xenograft mouse model","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP confirms m6A on ACSL3 mRNA, rescue experiment validates hierarchy, in vivo confirmation, single lab","pmids":["39833962"],"is_preprint":false},{"year":2025,"finding":"NT5DC2 interacts with ACSL3 and inhibits its ubiquitination, thereby stabilizing ACSL3 protein and suppressing ferroptosis in bladder cancer cells; silencing NT5DC2 abrogates oleic acid-mediated ACSL3 upregulation and increases ferroptosis.","method":"Co-immunoprecipitation for NT5DC2-ACSL3 interaction, ubiquitination assay, NT5DC2 knockdown, ACSL3 rescue experiment, ferroptosis assays, oleic acid treatment","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP for binding, ubiquitination assay for mechanism, rescue experiment, single lab","pmids":["41974665"],"is_preprint":false},{"year":2025,"finding":"PPARγ transcriptionally upregulates ACSL3 expression (confirmed by dual-luciferase reporter assay); the AngII-AT1R axis inhibits the PPARγ/ACSL3 pathway in hippocampal neurons, promoting ferroptosis and cognitive impairment under hypertensive conditions; ACSL3 overexpression alleviates AngII-induced neuronal ferroptosis.","method":"Dual-luciferase reporter assay for PPARγ binding to ACSL3 promoter, PPARγ agonist (rosiglitazone) treatment, ACSL3 overexpression, ferroptosis markers, behavioral tests in hypertensive rat model","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter assay confirms direct transcriptional regulation, combined with in vivo pharmacological and genetic approaches, single lab","pmids":["41933714"],"is_preprint":false},{"year":2026,"finding":"Genome-wide CRISPR loss-of-function screen identifies ACSL3 as a central determinant of hepatocyte susceptibility to palmitate-induced lipotoxicity; genetic deletion or pharmacological inhibition of ACSL3 renders hepatocytes resistant to palmitate-induced apoptosis and ER stress, reduces lipid droplet accumulation, and decreases saturated fatty acid incorporation into neutral lipids and phospholipids, blunting lipogenic programs.","method":"Genome-wide CRISPR-Cas9 screen, genetic ACSL3 deletion and pharmacological inhibition, isotope tracing for fatty acid incorporation into lipid classes, lipid droplet quantification, apoptosis/ER stress assays, human MASLD tissue analysis, single-cell and spatial transcriptomics","journal":"Hepatology communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — genome-wide CRISPR screen discovery validated by genetic KO and pharmacological inhibition with isotope tracing, multiple orthogonal methods, human tissue validation, single lab","pmids":["41564380"],"is_preprint":false},{"year":2024,"finding":"Sec14L6 directly interacts with ACSL3, and this interaction facilitates Sec14L6 targeting to lipid droplets and activates its PS transfer activity in vitro, linking ACSL3 to phospholipid transport for lipid droplet biogenesis.","method":"Co-immunoprecipitation for Sec14L6-ACSL3 interaction, in vitro PS transfer activity assay, Sec14L6 KO and rescue with lipid transfer-defective mutants, lipid droplet quantification","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct in vitro activity assay with Co-IP, preprint not yet peer-reviewed, single lab","pmids":["bio_10.1101_2024.10.20.619318"],"is_preprint":true}],"current_model":"ACSL3 is an ER/Golgi/lipid-droplet-associated long-chain acyl-CoA synthetase that activates fatty acids for incorporation into neutral lipids and phospholipids; it drives lipid droplet biogenesis, partitions fatty acids into β-oxidation or phospholipid pools (including phosphatidylinositols for prostaglandin synthesis via LPIAT1), mediates Golgi export of the Lyn kinase in a catalytic-activity-independent manner, participates in autophagosome formation, regulates ferroptosis susceptibility by modulating the MUFA/PUFA phospholipid ratio, and is controlled post-translationally by multiple ubiquitin E3 ligases (TRIM25, NEDD4, HRD1) and stabilized by SUMO2 and NT5DC2, while its transcription is directly regulated by LXR, MEF2D, PPARγ, and SP1, and its mRNA stability/splicing is modulated by m6A writers (METTL3, METTL7B) and the splicing factor HNRNPC."},"narrative":{"mechanistic_narrative":"ACSL3 is a long-chain acyl-CoA synthetase that activates fatty acids and channels them into distinct lipid pools, thereby governing lipid droplet biogenesis, membrane phospholipid composition, fatty acid oxidation versus storage decisions, and downstream cell-fate outcomes [PMID:17761945, PMID:36192773, PMID:41564380]. Through its enzymatic activity it partitions activated fatty acids into β-oxidation rather than triglyceride synthesis [PMID:17761945], drives accumulation of lipid droplets from exogenous fatty acids [PMID:36192773], and routes arachidonic acid into phosphatidylinositols to supply the LPIAT1-dependent prostaglandin pathway [PMID:32034305]. By shaping the monounsaturated-to-polyunsaturated phospholipid ratio it acts as a central determinant of ferroptosis susceptibility, with its enzymatic activity directly enhanced by vitamin A/ATRA to suppress lipid peroxidation [PMID:41909752]. ACSL3 also performs catalytic-activity-independent functions: it binds the open conformation of Lyn kinase to mediate its Golgi export to the plasma membrane [PMID:20605918] and is required for formation of fusion-competent autophagosomal membranes during starvation [PMID:40728409, PMID:41346954]. ACSL3 abundance is tightly set post-translationally by competing degradation and stabilization machinery — ubiquitin E3 ligases TRIM25, NEDD4, and HRD1 promote its turnover [PMID:39285846, PMID:39743027, PMID:41130543], whereas SUMO2 and NT5DC2 block its ubiquitination to stabilize it [PMID:40526170, PMID:41974665] — and transcriptionally by LXR, MEF2D, and PPARγ together with m6A- and splicing-dependent control of its mRNA [PMID:20219900, PMID:39744125, PMID:41933714, PMID:39160584, PMID:41037014]. As a hub linking fatty acid metabolism to cell survival, ACSL3 determines hepatocyte susceptibility to palmitate lipotoxicity [PMID:41564380] and modulates tumor growth, immune infiltration, and therapy resistance across multiple cancers [PMID:36192773, PMID:33127675, PMID:40059153].","teleology":[{"year":2007,"claim":"Established that ACSL3 is a regulated node controlling whether activated fatty acids enter oxidation or storage, defining its metabolic decision-making role.","evidence":"Transcriptional reporter, siRNA knockdown, and fatty acid oxidation/acyl-CoA synthetase assays in HepG2 cells and a hamster model under Oncostatin M stimulation","pmids":["17761945"],"confidence":"High","gaps":["Did not resolve which lipid species are preferentially generated","Mechanism by which ACSL3 activity biases toward β-oxidation not defined"]},{"year":2010,"claim":"Revealed that ACSL3 has a catalytic-activity-independent function in protein trafficking, distinct from its enzymatic role.","evidence":"Co-IP, conformation-dependent binding assays, domain-deletion mutants lacking the LR2 catalytic domain, and live-cell trafficking imaging of Lyn kinase Golgi export","pmids":["20605918"],"confidence":"High","gaps":["Structural basis of ACSL3-Lyn interaction not solved","Whether ACSL3 traffics other kinases unknown"]},{"year":2010,"claim":"Identified direct transcriptional control of ACSL3 by LXR, linking nuclear lipid sensing to ACSL3-dependent fatty acid uptake.","evidence":"Promoter reporter assays with LXR response element mapping, siRNA knockdown, and acyl-CoA synthetase/fatty acid uptake assays in placental trophoblasts","pmids":["20219900"],"confidence":"High","gaps":["Tissue-specificity of LXR regulation not addressed","Interplay with other transcriptional inputs unexplored"]},{"year":2017,"claim":"Localized ACSL3 function to insulin secretory granules and showed substrate selectivity toward arachidonate, connecting it to regulated secretion.","evidence":"Subcellular fractionation, stable shRNA knockdown, glucose-stimulated insulin secretion in INS-1 cells and human islets, and phospholipid profiling","pmids":["28193492"],"confidence":"High","gaps":["Molecular link between acyl-CoA synthesis and secretion not defined","Whether granule targeting requires specific partners unknown"]},{"year":2018,"claim":"Defined ACSL3's distinct subcellular itinerary (trans-Golgi/endosomes and lipid droplets), separating it spatially from the ER-resident ACSL4.","evidence":"Subcellular fractionation, confocal immunofluorescence, and tumor array immunohistochemistry in fibrosarcoma and breast cancer cells","pmids":["29450800"],"confidence":"Medium","gaps":["Targeting determinants for Golgi/LD localization not mapped","Functional consequence of compartment-specific localization not tested"]},{"year":2020,"claim":"Showed ACSL3 supplies arachidonate-derived phosphatidylinositols to an LPIAT1 axis sustaining prostaglandin synthesis, a tumor-promoting lipid signaling output.","evidence":"ACSL3/LPIAT1 knockdown in lung cancer lines and KrasG12D mice, lipidomics, prostaglandin measurement, and in vivo tumorigenesis assays","pmids":["32034305"],"confidence":"High","gaps":["Direct enzymatic flux of arachidonate through ACSL3 to PI not isotopically traced","Generalizability beyond lung cancer not established"]},{"year":2020,"claim":"Connected ACSL3 to autophagy machinery via direct binding to GABARAPL2 and ER recruitment of the UFM1 conjugation system.","evidence":"CRISPR endogenous ATG8 tagging, affinity purification-MS interactome, Co-IP, and knockdown of ufmylation/ER-phagy components","pmids":["32843575"],"confidence":"High","gaps":["Whether ACSL3 LIR-mediated binding requires lipid catalysis unclear","Physiological role of ACSL3-UFM1 link in vivo not tested"]},{"year":2020,"claim":"Demonstrated ACSL3 dependence for lipid droplet accumulation from exogenous fatty acids and identified its role in shaping ferroptosis susceptibility in cancer.","evidence":"Genetic/pharmacologic ACSL3 inhibition, isotope-tracing lipidomics, orthotopic ccRCC mouse model, and FACS ferroptosis assays","pmids":["36192773"],"confidence":"High","gaps":["Lipid species mediating ferroptosis modulation not fully resolved","Dependence on fatty acid composition mechanistically incomplete"]},{"year":2020,"claim":"Established an ACSL3-PAI-1 axis by which ACSL3 reshapes the tumor immune microenvironment in pancreatic cancer.","evidence":"Acsl3 genetic KO in mouse PDAC, in vivo tumor growth, immune infiltrate flow cytometry, and PAI-1 measurement/inhibition with therapy response","pmids":["33127675"],"confidence":"High","gaps":["How ACSL3 metabolic activity controls PAI-1 secretion is unclear","Whether the effect is fully lipid-dependent untested"]},{"year":2021,"claim":"Placed ACSL3 within a Rab18-PLIN2 lipid droplet complex regulating triacylglycerol levels and LD dynamics.","evidence":"Co-IP, Rab18 overexpression/knockdown, lipid droplet staining, and TAG quantification in C2C12 myoblasts","pmids":["33713834"],"confidence":"Medium","gaps":["Single primary binding method without reciprocal structural validation","Direct vs indirect nature of complex assembly unresolved"]},{"year":2024,"claim":"Resolved post-translational control of ACSL3 stability, showing E3 ligase TRIM25 (recruited by ANKRD1) drives K63-ubiquitination and degradation to amplify ferroptosis.","evidence":"IP-MS, reciprocal Co-IP, proximity ligation, K63-linkage ubiquitination assays, and in vivo rAAV9 knockdown in renal ischemia-reperfusion","pmids":["39285846"],"confidence":"High","gaps":["Whether K63-ubiquitination targets ACSL3 to proteasome or another fate unclear","Other adaptors recruiting TRIM25 to ACSL3 unknown"]},{"year":2025,"claim":"Expanded the ACSL3 degradation network to NEDD4 (via TNFAIP3) and HRD1, defining multiple converging ubiquitin pathways that tune ACSL3 levels in disease.","evidence":"Co-IP, ubiquitination assays identifying E3 ligases, and in vivo AAV knockdown in TBI and ethanol-diet liver injury models","pmids":["39743027","41130543"],"confidence":"High","gaps":["Relative contribution of each E3 ligase across tissues not compared","Ubiquitination sites on ACSL3 not mapped"]},{"year":2025,"claim":"Identified stabilizing factors SUMO2 and NT5DC2 that block ACSL3 ubiquitination, establishing a balance between degradation and protection that sets ferroptosis sensitivity.","evidence":"Co-IP, ubiquitination assays, and ACSL3 rescue/knockdown ferroptosis experiments in hepatocellular and bladder cancer cells","pmids":["40526170","41974665"],"confidence":"Medium","gaps":["Whether SUMO2 acts via direct SUMOylation of ACSL3 not resolved","Single-lab Co-IP evidence per partner without structural validation"]},{"year":2025,"claim":"Demonstrated that ACSL3 enzymatic activity directly governs the MUFA/PUFA phospholipid ratio and can be pharmacologically enhanced (vitamin A/ATRA) to suppress ferroptosis and extend lifespan.","evidence":"In vitro enzymatic activity/binding assays, phospholipid lipidomics, ferroptosis assays, and ACSL3-dependent C. elegans lifespan analysis","pmids":["41909752"],"confidence":"High","gaps":["Structural basis of vitamin A binding to ACSL3 not solved","Mammalian lifespan/healthspan effects not tested"]},{"year":2025,"claim":"Refined ACSL3's catalytic-activity-independent role in autophagy to an early step of fusion-competent autophagosomal membrane formation, regulated by SYNTAXIN17.","evidence":"Knockdown/overexpression with enzymatic-dead mutants, WIPI2/LC3/FIP200 imaging, LC3 lipidation assays, and SYNTAXIN17 epistasis","pmids":["40728409","41346954"],"confidence":"High","gaps":["Molecular mechanism by which ACSL3 confers membrane fusion competence unknown","How ACSL3 partitions between LD biogenesis and autophagosome roles unclear"]},{"year":2026,"claim":"Positioned ACSL3 as a central genome-wide determinant of hepatocyte palmitate lipotoxicity, integrating its substrate routing into a tractable therapeutic node.","evidence":"Genome-wide CRISPR screen with genetic/pharmacologic validation, isotope tracing of fatty acid incorporation, and human MASLD tissue with spatial transcriptomics","pmids":["41564380"],"confidence":"High","gaps":["Whether catalytic vs non-catalytic functions drive lipotoxicity not dissected","Long-term consequences of ACSL3 inhibition in liver not established"]},{"year":null,"claim":"How ACSL3 mechanistically toggles between its catalytic functions (fatty acid activation, phospholipid remodeling, ferroptosis control) and its catalytic-activity-independent functions (Lyn export, autophagosome membrane formation) within a single protein remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model coupling catalytic and scaffolding activities","Determinants directing ACSL3 to Golgi vs lipid droplet vs autophagosomal membranes unknown","Ubiquitination/SUMOylation site mapping incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,2,26]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,9,26]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,24]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,8]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[8,10,13]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[11,22]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,10,32]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell 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B","url":"https://pubmed.ncbi.nlm.nih.gov/41909752","citation_count":1,"is_preprint":false},{"pmid":"39584470","id":"PMC_39584470","title":"Core promoter identification and transcriptional regulation of porcine ACSL3 gene.","date":"2024","source":"Animal biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/39584470","citation_count":0,"is_preprint":false},{"pmid":"41346954","id":"PMC_41346954","title":"Involvement of ACSL3 in the formation of autophagosomes and lipid droplets during starvation conditions.","date":"2025","source":"Autophagy reports","url":"https://pubmed.ncbi.nlm.nih.gov/41346954","citation_count":0,"is_preprint":false},{"pmid":"41564380","id":"PMC_41564380","title":"Genome-wide CRISPR screen identifies ACSL3 as a regulator of lipotoxicity and progression of MASLD.","date":"2026","source":"Hepatology communications","url":"https://pubmed.ncbi.nlm.nih.gov/41564380","citation_count":0,"is_preprint":false},{"pmid":"41130543","id":"PMC_41130543","title":"HRD1 promotes chronic alcoholic liver disease by mediating ACSL3 ubiquitination and degradation.","date":"2025","source":"Toxicology letters","url":"https://pubmed.ncbi.nlm.nih.gov/41130543","citation_count":0,"is_preprint":false},{"pmid":"41974665","id":"PMC_41974665","title":"NT5DC2 inhibits ferroptosis by stabilizing ACSL3 in bladder cancer.","date":"2026","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/41974665","citation_count":0,"is_preprint":false},{"pmid":"41037014","id":"PMC_41037014","title":"HNRNPC aggravates the symptoms of preeclampsia by regulating m6A-dependent alternative splicing of ACSL3.","date":"2025","source":"Journal of hypertension","url":"https://pubmed.ncbi.nlm.nih.gov/41037014","citation_count":0,"is_preprint":false},{"pmid":"41933714","id":"PMC_41933714","title":"AngII promotes hippocampal neuron ferroptosis via inhibiting the PPARγ/ACSL3 axis leading to hypertension-related cognitive impairment.","date":"2026","source":"Experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/41933714","citation_count":0,"is_preprint":false},{"pmid":"41301966","id":"PMC_41301966","title":"Oleic Acid Improves Goat Sperm Quality by Enhancing the MBOAT2/ACSL3 Pathway to Attenuate Ferroptosis.","date":"2025","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/41301966","citation_count":0,"is_preprint":false},{"pmid":"42002520","id":"PMC_42002520","title":"Vitamin D Mitigates Klebsiella pneumoniae-Induced Pneumonia by Regulating Macrophage Polarization Through miR-223/ACSL3 Axis-Mediated Lipid Metabolism Reprogramming.","date":"2026","source":"The journal of gene medicine","url":"https://pubmed.ncbi.nlm.nih.gov/42002520","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.08.25335383","title":"Identification of Key Genes Governing the Effects of Physical Activity on Ferroptosis in Alzheimer’s Disease Patients: A Machine Learning-Based Study","date":"2025-09-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.08.25335383","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.20.619318","title":"Sec14L6 is a PS and PI4P transporter that promotes lipid droplet formation","date":"2024-10-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.20.619318","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.28.655990","title":"Genetic regulation of fatty acid content in adipose tissue","date":"2025-05-29","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.28.655990","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":31500,"output_tokens":9139,"usd":0.115793,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19453,"output_tokens":5288,"usd":0.114733,"stage2_stop_reason":"end_turn"},"total_usd":0.230526,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"ACSL3 associates with the C-lobe of the Lyn kinase domain on the Golgi in a conformation-dependent manner (open conformation of Lyn required) and mediates Golgi export of Lyn to the plasma membrane; an ACSL3 mutant lacking the LR2 catalytic domain retains both Lyn-binding and Golgi export activity, indicating the function is independent of ACSL3 enzymatic activity.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and siRNA knockdown of ACSL3 with live-cell trafficking assays, domain-deletion mutagenesis, confocal imaging\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays, functional domain mutagenesis, overexpression and knockdown with defined trafficking phenotype, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"20605918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Oncostatin M activates transcription of ACSL3 (and ACSL5) in hepatocytes through the ERK signaling pathway; increased ACSL3 activity partitions fatty acids into β-oxidation rather than triglyceride synthesis, reducing TG accumulation; siRNA knockdown of ACSL3/ACSL5 abrogates OM-enhanced fatty acid oxidation.\",\n      \"method\": \"Transcriptional reporter assays, siRNA knockdown, fatty acid oxidation assays in HepG2 cells and in vivo hamster model, acyl-CoA synthetase activity measurement\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA rescue, enzymatic activity assay, in vivo validation, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"17761945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"LXR activation directly regulates ACSL3 transcription through a conserved LXR response element in the ACSL3 promoter in human placental trophoblast cells, increasing acyl-CoA synthetase activity and fatty acid uptake; ACSL3 silencing attenuates LXR-mediated increases in acyl-CoA synthetase activity.\",\n      \"method\": \"Promoter reporter assays with LXR response element identification, siRNA knockdown, acyl-CoA synthetase activity assay, fatty acid uptake assay\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — promoter mutagenesis/reporter assay combined with siRNA knockdown and enzymatic activity measurement, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"20219900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ACSL3 expression is induced by ER stress (tunicamycin) in hepatocytes; ACSL3 shRNA (but not ACSL1 shRNA) blocks ER stress-induced lipid accumulation; GSK-3β acts upstream of ACSL3 in this pathway, as GSK-3β inhibitors or shRNA suppress ACSL3 upregulation and lipid accumulation.\",\n      \"method\": \"shRNA knockdown, GSK-3β inhibitor treatment, lipid accumulation assays (Oil Red O), western blot in HuH-7 and HepG2 cells, hepatitis B virus mutant large surface protein model\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic (shRNA) and pharmacological dissection of pathway order, single lab, two orthogonal approaches\",\n      \"pmids\": [\"21328461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ACSL3 mediates palmitic acid (PA)-induced osteoblastic differentiation and calcium deposition in vascular smooth muscle cells; ACS inhibitor or ACSL3 siRNA prevents PA-induced BMP-2 and Msx2 expression and calcium deposition; adenovirus-mediated ACSL3 overexpression enhances these effects; EPA inhibits ACSL3 expression and downstream osteoblastic gene induction.\",\n      \"method\": \"siRNA knockdown, adenovirus-mediated overexpression, ACS pharmacological inhibitor, qPCR for osteoblastic markers, calcium deposition assay, immunohistochemistry of human plaques\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic (siRNA) and gain-of-function (adenovirus overexpression) plus pharmacological inhibition with defined molecular phenotype, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"23840832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ACSL3 overexpression in androgen-dependent LNCaP prostate cancer cells upregulates AKR1C3 (involved in steroidogenesis converting DHEAS to testosterone) and downregulates the androgen-inactivating enzyme UGT2B, promoting intratumoral androgen synthesis and cell proliferation in response to DHEAS.\",\n      \"method\": \"ACSL3 overexpression in LNCaP cells, gene expression profiling, testosterone measurement by mass spectrometry, cell proliferation assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — overexpression with defined downstream gene expression and metabolite measurement, single lab, single main approach\",\n      \"pmids\": [\"28771887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ACSL3 and ACSL4 are concentrated in insulin secretory granules of pancreatic beta cells; shRNA-mediated knockdown of ACSL3 or ACSL4 inhibits glucose-stimulated insulin secretion ~50% in INS-1 832/13 cells and in human pancreatic islets; ACSL3 knockdown preferentially reduces arachidonate over palmitate as substrate.\",\n      \"method\": \"Subcellular fractionation, immunostaining, stable shRNA knockdown cell lines, glucose-stimulated insulin secretion assay, ACSL enzyme activity assay, phospholipid profiling\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation, stable knockdown with functional assay validated in human islets, enzymatic activity confirmation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28193492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Upper small intestinal ACSL3 expression is required for fatty acid-dependent pre-absorptive signaling that regulates glucose homeostasis; high-fat feeding reduces ACSL3 expression and impairs fatty acid sensing; restoration of Lactobacillus gasseri increases ACSL3 expression and restores fatty acid sensing and glucose tolerance in rodents.\",\n      \"method\": \"Upper small intestinal infusion of lipids, surgical duodenal cannulation, in vivo glucose clamps, ACSL3 knockdown/expression measurements, microbiota transplantation, L. gasseri probiotic administration\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo glucose clamp measurements with genetic pathway dissection, microbiota manipulation with defined molecular readout, multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"29514066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Endogenous ACSL3 in fibrosarcoma and breast cancer cells localizes to the trans-Golgi network/endosomal compartments, distinct from ACSL4 which follows the endoplasmic reticulum pattern; both isoforms associate with lipid droplets.\",\n      \"method\": \"Subcellular fractionation, confocal immunofluorescence imaging, immunohistochemistry of tumor arrays\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation combined with confocal imaging, replicated across two cell lines, single lab\",\n      \"pmids\": [\"29450800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ACSL3 channels arachidonic acid (AA) into phosphatidylinositols, providing a substrate pool for LPIAT1 to sustain elevated prostaglandin synthesis in non-small cell lung cancer; LPIAT1 knockdown suppresses proliferation and in vivo tumorigenesis, defining an ACSL3-LPIAT1 axis for prostaglandin production.\",\n      \"method\": \"ACSL3 and LPIAT1 knockdown in lung cancer cell lines and KrasG12D mouse models, lipidomics, prostaglandin measurement, proliferation and anchorage-independent growth assays, in vivo tumorigenesis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockdown validated in cell lines and clinically relevant in vivo mouse model with lipidomic substrate tracing, multiple orthogonal methods, replicated across model systems\",\n      \"pmids\": [\"32034305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ACSL3 is required for lipid droplet accumulation from exogenous serum-derived fatty acids (not de novo lipogenesis) in clear cell renal cell carcinoma cells; genetic or pharmacologic ACSL3 suppression is cytotoxic to ccRCC in vitro and reduces tumor weight in an orthotopic mouse model; ACSL3 inhibition decreases ferroptosis susceptibility in a manner dependent on exogenous fatty acid composition.\",\n      \"method\": \"siRNA/shRNA knockdown, pharmacological inhibition, isotope-tracing lipidomics, Oil Red O staining, cell viability assays, orthotopic mouse tumor model, FACS-based ferroptosis assays\",\n      \"journal\": \"Cancer & metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological inhibition with isotope tracing, in vitro and in vivo validation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"36192773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ACSL3 is a direct binding partner of GABARAPL2 (via LC3-interacting regions); through this interaction GABARAPL2 is recruited to the ER, anchoring UBA5 (UFM1-activating enzyme) at the ER; ACSL3 depletion and lipid droplet induction affect abundance of ufmylation components and ER-phagy, establishing ACSL3 as a regulator of the UFM1 conjugation pathway.\",\n      \"method\": \"CRISPR/Cas9 endogenous tagging of ATG8 proteins, interaction proteomics (affinity purification–mass spectrometry), co-immunoprecipitation, knockdown experiments\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — endogenous protein tagging by CRISPR, affinity purification–MS interactome with Co-IP validation, functional knockdown, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"32843575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ACSL3 knockout in pancreatic ductal adenocarcinoma hinders tumor progression, reduces tumor fibrosis, reduces immunosuppressive cell infiltration, and increases cytotoxic T cell infiltration; this is mediated at least in part through decreased PAI-1 secretion from tumor cells, defining an ACSL3–PAI-1 signaling axis.\",\n      \"method\": \"Acsl3 genetic knockout in mouse PDAC models, in vivo tumor growth assays, flow cytometry of tumor-infiltrating immune cells, PAI-1 measurement, PAI-1 pharmacological inhibition with chemo/immunotherapy response assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO in vivo with multiple defined phenotypic readouts (fibrosis, immunity, PAI-1 secretion), pharmacological rescue experiment, single lab but rigorous in vivo model\",\n      \"pmids\": [\"33127675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rab18 interacts with ACSL3 on lipid droplets and promotes ACSL3 LD localization; Rab18 also binds PLIN2, which recruits Rab18 from ER to LDs; the Rab18-PLIN2-ACSL3 complex regulates triacylglycerol levels and lipid droplet dynamics in myoblast cells.\",\n      \"method\": \"Co-immunoprecipitation, Rab18 overexpression and knockdown, lipid droplet staining, TAG quantification, confocal imaging in C2C12 cells\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular and cell biology of lipids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP binding shown, overexpression and loss-of-function with defined phenotype, single lab, single main binding method\",\n      \"pmids\": [\"33713834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MAT2A mediates ferroptosis resistance in gastric cancer by producing S-adenosylmethionine (SAM), which upregulates ACSL3 expression via H3K4me3 trimethylation at the ACSL3 promoter, thereby increasing resistance to ferroptosis.\",\n      \"method\": \"Pharmacological and genetic blockade of methionine cycle, chromatin immunoprecipitation (H3K4me3 at ACSL3 promoter), gene expression analysis, in vitro and in vivo ferroptosis assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for H3K4me3 at ACSL3 promoter combined with genetic/pharmacologic methionine cycle blockade and ferroptosis phenotype, single lab, two orthogonal methods\",\n      \"pmids\": [\"35182729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"BRD4 controls the splicing efficiency of ACSL3 pre-mRNA by recruiting SRPK2 to assemble a splicing catalytic platform; the AMP-binding domain of ACSL3 influences arachidonic acid synthesis and thus determines susceptibility to erastin-induced ferroptosis in osteosarcoma cells.\",\n      \"method\": \"BRD4 inhibition (in vitro and in vivo), SRPK2 co-IP, RT-PCR splicing analysis, ACSL3 domain mutants, arachidonic acid measurement, ferroptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — splicing analysis combined with domain mutagenesis and functional ferroptosis assay, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"37993451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FTO demethylates m6A modifications on ACSL3 mRNA (and GPX4 mRNA), decreasing their stability and expression, thereby sensitizing oral squamous cell carcinoma cells to ferroptosis in vitro and in vivo.\",\n      \"method\": \"FTO overexpression/knockdown, m6A methylation analysis (MeRIP), mRNA stability assays, ferroptosis assays (lipid ROS, cell viability) in vitro and in vivo\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP for m6A site identification combined with mRNA stability assays and functional ferroptosis readout in vitro and in vivo, single lab\",\n      \"pmids\": [\"38003537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ANKRD1 directly binds ACSL3 and promotes its degradation via K63-linked ubiquitination catalyzed by the E3 ligase TRIM25, reducing ACSL3 protein levels, amplifying lipid peroxidation and ferroptosis, and exacerbating renal ischemia-reperfusion injury.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry to identify ANKRD1 interactors, Co-IP and proximity ligation assay for ANKRD1-ACSL3 and TRIM25-ACSL3 interactions, ubiquitination assays (K63-linkage), ANKRD1 knockdown (rAAV9) in vivo, siRNA in vitro, cell viability and lipid peroxidation assays\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS interactome, reciprocal Co-IP, ubiquitination assay with linkage specificity, in vivo genetic KD with defined phenotype, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"39285846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MEF2D directly binds the promoter region of ACSL3 and transcriptionally upregulates ACSL3 expression, inhibiting ferroptosis and enhancing sorafenib resistance in hepatocellular carcinoma.\",\n      \"method\": \"Promoter binding assays (ChIP or EMSA), gene expression analysis, ACSL3 silencing in sorafenib-resistant HCC cells, ferroptosis level assessment\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — promoter binding assay plus functional knockdown, single lab, single binding approach\",\n      \"pmids\": [\"39744125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL3 in cancer-associated fibroblast-derived exosomes induces m6A modification on ACSL3 mRNA, stabilizing ACSL3 expression, which promotes colorectal cancer cell proliferation, metastasis, and suppresses ferroptosis; METTL3 knockdown in CAFs reverses these effects and is rescued by ACSL3 overexpression.\",\n      \"method\": \"Methylated RNA immunoprecipitation (MeRIP), dual-luciferase reporter assay, exosome isolation, METTL3 knockdown in CAFs, ACSL3 overexpression rescue, in vitro and in vivo tumor models\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP confirms m6A site, rescue experiment validates mechanistic hierarchy, in vivo confirmation, single lab\",\n      \"pmids\": [\"39160584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACSL3 interacts with YES1 (Src-family kinase) and suppresses its activation (phospho-Tyr419), consequently inhibiting YAP1 nuclear colocalization and transcriptional complex formation in breast cancer cells; ACSL3 knockdown promotes cell proliferation, migration, and EMT.\",\n      \"method\": \"Co-immunoprecipitation for ACSL3-YES1 interaction, phospho-YES1 measurement, ACSL3 knockdown/overexpression, YAP1 nuclear localization assay, in vitro and in vivo functional assays\",\n      \"journal\": \"Cancer biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP for ACSL3-YES1 binding plus phosphorylation measurement and defined downstream signaling phenotype, single lab\",\n      \"pmids\": [\"38953696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TNFAIP3 promotes ACSL3 degradation via NEDD4-mediated ubiquitination, reducing ACSL3 levels, enhancing lipid peroxidation and ferroptosis in neurons after traumatic brain injury; TNFAIP3 overexpression increases neuronal cell death, while TNFAIP3 knockdown (AAV-shTNFAIP3) alleviates ferroptosis and cognitive impairment.\",\n      \"method\": \"Co-IP for TNFAIP3-ACSL3 interaction, ubiquitination assay with NEDD4, TNFAIP3 overexpression/knockdown, AAV-shTNFAIP3 in mouse TBI model, lipid peroxidation and ferroptosis markers\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP combined with ubiquitination assay identifying the E3 ligase and in vivo rescue, single lab\",\n      \"pmids\": [\"39743027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HRD1 (an ER-associated E3 ubiquitin ligase) ubiquitinates ACSL3 and promotes its proteasomal degradation; HRD1 knockdown increases ACSL3 levels, suppresses fatty acid synthesis, promotes fatty acid oxidation, and alleviates alcohol-induced hepatic injury and steatosis.\",\n      \"method\": \"AAV9-shRNA knockdown of HRD1 and ACSL3 in mice, siRNA in HepG2 cells, co-immunoprecipitation for HRD1-ACSL3 interaction, ubiquitination assay, lipid metabolism measurements, Lieber-DeCarli ethanol diet model\",\n      \"journal\": \"Toxicology letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP combined with ubiquitination assay plus in vivo genetic knockdown with rescue phenotype, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"41130543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SUMO2 directly binds ACSL3 and inhibits its entry into the ubiquitin-proteasome degradation pathway, stabilizing ACSL3 protein and thereby suppressing ferroptosis in hepatocellular carcinoma cells; ACSL3 knockdown in SUMO2-overexpressing cells reverses SUMO2's anti-ferroptotic effect.\",\n      \"method\": \"Co-immunoprecipitation for SUMO2-ACSL3 interaction, ACSL3 ubiquitination assay, SUMO2 overexpression/knockdown, ACSL3 rescue knockdown, ferroptosis marker assays\",\n      \"journal\": \"Discover oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP for binding, ubiquitination assay for mechanism, rescue experiment for pathway order, single lab\",\n      \"pmids\": [\"40526170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ACSL3 knockdown impairs starvation-induced autophagy and causes formation of enlarged autophagosome-like structures negative for WIPI2; ACSL3 overexpression induces WIPI2-positive but LC3-negative dots under normal nutrition; both effects are independent of ACSL3 enzymatic activity, suggesting ACSL3 functions in formation of fusion-competent autophagosomal membranes at a stage distinct from ACSL4.\",\n      \"method\": \"Knockdown and overexpression of ACSL3 and ACSL4, autophagy induction by starvation, immunofluorescence for WIPI2, LC3, FIP200, LC3 lipidation assay, enzymatic activity-dead mutants\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown and gain-of-function with domain/activity mutants and multiple autophagy marker readouts, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40728409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ACSL3 is required for lipid droplet biogenesis during starvation and for formation of functional autophagosomes; under starvation ACSL3 is regulated by SYNTAXIN17; ACSL3 functions at an early autophagy stage (formation of autophagosomes) independently of its enzymatic activity.\",\n      \"method\": \"Knockdown of ACSL3, immunofluorescence for autophagy markers (FIP200, WIPI2, LC3), lipid droplet staining, genetic epistasis with SYNTAXIN17\",\n      \"journal\": \"Autophagy reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — knockdown with multiple marker readouts, single lab, overlap with companion paper (PMID 40728409)\",\n      \"pmids\": [\"41346954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Vitamin A and ATRA directly target ACSL3 and enhance its enzymatic activity; this ACSL3-dependent mechanism increases the MUFA/PUFA ratio in phospholipids, preventing lipid peroxidation and suppressing ferroptosis; vitamin A and its analogue D3 extend C. elegans lifespan in an ACSL3-dependent manner.\",\n      \"method\": \"Biochemical binding and enzymatic activity assays, phospholipid lipidomics (MUFA/PUFA ratio), ferroptosis assays, VA analogue structure-activity relationship, C. elegans lifespan assay with ACSL3 genetic dependence\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic activity assay with drug binding, lipidomics confirming substrate mechanism, in vivo genetic dependence assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"41909752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ACSL3 promotes synthesis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which activates the PPARα pathway and enhances transcription of downstream lipid metabolism genes, promoting hepatocellular carcinoma growth and metastasis by accelerating lipid catabolism and anabolism.\",\n      \"method\": \"Proteomic and scRNA-seq analysis, ACSL3 siRNA/nanoparticle-mediated knockdown, lipidomics (POPC measurement), PPARα pathway reporter assays, in vitro and in vivo tumor models\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lipidomics identifies specific lipid product combined with pathway reporter assay and in vivo genetic knockdown, single lab\",\n      \"pmids\": [\"40059153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HNRNPC binds to ACSL3 RNA and promotes exon 10 skipping (alternative splicing), generating a short ACSL3-S isoform; m6A modification at the ACSL3 mRNA enhances HNRNPC binding; FBXW11 acts as an E3 ubiquitin ligase to ubiquitinate and degrade HNRNPC; HNRNPC knockdown in mice alleviates preeclampsia symptoms and dysregulates ferroptosis markers.\",\n      \"method\": \"RT-PCR and RT-qPCR for splicing analysis, co-IP for HNRNPC-ACSL3 RNA binding, m6A site mutation, in vitro ubiquitination assay, FBXW11 co-IP, mouse preeclampsia model with HNRNPC knockdown\",\n      \"journal\": \"Journal of hypertension\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — splicing assay with m6A mutation to confirm binding, in vitro ubiquitination, in vivo phenotype, multiple methods, single lab\",\n      \"pmids\": [\"41037014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL7B promotes m6A modification on ACSL3 mRNA, stabilizing its expression and inhibiting erastin-induced ferroptosis in bladder cancer cells; METTL7B knockdown reduces ACSL3 protein levels and induces ferroptosis; ACSL3 overexpression rescues the pro-ferroptotic effect of METTL7B knockdown.\",\n      \"method\": \"MeRIP for m6A modification, METTL7B knockdown/overexpression, ACSL3 expression analysis, ferroptosis assays (lipid ROS, Fe2+, MDA), ACSL3 rescue experiment, xenograft mouse model\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP confirms m6A on ACSL3 mRNA, rescue experiment validates hierarchy, in vivo confirmation, single lab\",\n      \"pmids\": [\"39833962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NT5DC2 interacts with ACSL3 and inhibits its ubiquitination, thereby stabilizing ACSL3 protein and suppressing ferroptosis in bladder cancer cells; silencing NT5DC2 abrogates oleic acid-mediated ACSL3 upregulation and increases ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation for NT5DC2-ACSL3 interaction, ubiquitination assay, NT5DC2 knockdown, ACSL3 rescue experiment, ferroptosis assays, oleic acid treatment\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP for binding, ubiquitination assay for mechanism, rescue experiment, single lab\",\n      \"pmids\": [\"41974665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PPARγ transcriptionally upregulates ACSL3 expression (confirmed by dual-luciferase reporter assay); the AngII-AT1R axis inhibits the PPARγ/ACSL3 pathway in hippocampal neurons, promoting ferroptosis and cognitive impairment under hypertensive conditions; ACSL3 overexpression alleviates AngII-induced neuronal ferroptosis.\",\n      \"method\": \"Dual-luciferase reporter assay for PPARγ binding to ACSL3 promoter, PPARγ agonist (rosiglitazone) treatment, ACSL3 overexpression, ferroptosis markers, behavioral tests in hypertensive rat model\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter assay confirms direct transcriptional regulation, combined with in vivo pharmacological and genetic approaches, single lab\",\n      \"pmids\": [\"41933714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Genome-wide CRISPR loss-of-function screen identifies ACSL3 as a central determinant of hepatocyte susceptibility to palmitate-induced lipotoxicity; genetic deletion or pharmacological inhibition of ACSL3 renders hepatocytes resistant to palmitate-induced apoptosis and ER stress, reduces lipid droplet accumulation, and decreases saturated fatty acid incorporation into neutral lipids and phospholipids, blunting lipogenic programs.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screen, genetic ACSL3 deletion and pharmacological inhibition, isotope tracing for fatty acid incorporation into lipid classes, lipid droplet quantification, apoptosis/ER stress assays, human MASLD tissue analysis, single-cell and spatial transcriptomics\",\n      \"journal\": \"Hepatology communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — genome-wide CRISPR screen discovery validated by genetic KO and pharmacological inhibition with isotope tracing, multiple orthogonal methods, human tissue validation, single lab\",\n      \"pmids\": [\"41564380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Sec14L6 directly interacts with ACSL3, and this interaction facilitates Sec14L6 targeting to lipid droplets and activates its PS transfer activity in vitro, linking ACSL3 to phospholipid transport for lipid droplet biogenesis.\",\n      \"method\": \"Co-immunoprecipitation for Sec14L6-ACSL3 interaction, in vitro PS transfer activity assay, Sec14L6 KO and rescue with lipid transfer-defective mutants, lipid droplet quantification\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct in vitro activity assay with Co-IP, preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [\"bio_10.1101_2024.10.20.619318\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ACSL3 is an ER/Golgi/lipid-droplet-associated long-chain acyl-CoA synthetase that activates fatty acids for incorporation into neutral lipids and phospholipids; it drives lipid droplet biogenesis, partitions fatty acids into β-oxidation or phospholipid pools (including phosphatidylinositols for prostaglandin synthesis via LPIAT1), mediates Golgi export of the Lyn kinase in a catalytic-activity-independent manner, participates in autophagosome formation, regulates ferroptosis susceptibility by modulating the MUFA/PUFA phospholipid ratio, and is controlled post-translationally by multiple ubiquitin E3 ligases (TRIM25, NEDD4, HRD1) and stabilized by SUMO2 and NT5DC2, while its transcription is directly regulated by LXR, MEF2D, PPARγ, and SP1, and its mRNA stability/splicing is modulated by m6A writers (METTL3, METTL7B) and the splicing factor HNRNPC.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACSL3 is a long-chain acyl-CoA synthetase that activates fatty acids and channels them into distinct lipid pools, thereby governing lipid droplet biogenesis, membrane phospholipid composition, fatty acid oxidation versus storage decisions, and downstream cell-fate outcomes [#1, #10, #32]. Through its enzymatic activity it partitions activated fatty acids into β-oxidation rather than triglyceride synthesis [#1], drives accumulation of lipid droplets from exogenous fatty acids [#10], and routes arachidonic acid into phosphatidylinositols to supply the LPIAT1-dependent prostaglandin pathway [#9]. By shaping the monounsaturated-to-polyunsaturated phospholipid ratio it acts as a central determinant of ferroptosis susceptibility, with its enzymatic activity directly enhanced by vitamin A/ATRA to suppress lipid peroxidation [#26]. ACSL3 also performs catalytic-activity-independent functions: it binds the open conformation of Lyn kinase to mediate its Golgi export to the plasma membrane [#0] and is required for formation of fusion-competent autophagosomal membranes during starvation [#24, #25]. ACSL3 abundance is tightly set post-translationally by competing degradation and stabilization machinery — ubiquitin E3 ligases TRIM25, NEDD4, and HRD1 promote its turnover [#17, #21, #22], whereas SUMO2 and NT5DC2 block its ubiquitination to stabilize it [#23, #30] — and transcriptionally by LXR, MEF2D, and PPARγ together with m6A- and splicing-dependent control of its mRNA [#2, #18, #31, #19, #28]. As a hub linking fatty acid metabolism to cell survival, ACSL3 determines hepatocyte susceptibility to palmitate lipotoxicity [#32] and modulates tumor growth, immune infiltration, and therapy resistance across multiple cancers [#10, #12, #27].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that ACSL3 is a regulated node controlling whether activated fatty acids enter oxidation or storage, defining its metabolic decision-making role.\",\n      \"evidence\": \"Transcriptional reporter, siRNA knockdown, and fatty acid oxidation/acyl-CoA synthetase assays in HepG2 cells and a hamster model under Oncostatin M stimulation\",\n      \"pmids\": [\"17761945\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which lipid species are preferentially generated\", \"Mechanism by which ACSL3 activity biases toward β-oxidation not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed that ACSL3 has a catalytic-activity-independent function in protein trafficking, distinct from its enzymatic role.\",\n      \"evidence\": \"Co-IP, conformation-dependent binding assays, domain-deletion mutants lacking the LR2 catalytic domain, and live-cell trafficking imaging of Lyn kinase Golgi export\",\n      \"pmids\": [\"20605918\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ACSL3-Lyn interaction not solved\", \"Whether ACSL3 traffics other kinases unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified direct transcriptional control of ACSL3 by LXR, linking nuclear lipid sensing to ACSL3-dependent fatty acid uptake.\",\n      \"evidence\": \"Promoter reporter assays with LXR response element mapping, siRNA knockdown, and acyl-CoA synthetase/fatty acid uptake assays in placental trophoblasts\",\n      \"pmids\": [\"20219900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specificity of LXR regulation not addressed\", \"Interplay with other transcriptional inputs unexplored\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Localized ACSL3 function to insulin secretory granules and showed substrate selectivity toward arachidonate, connecting it to regulated secretion.\",\n      \"evidence\": \"Subcellular fractionation, stable shRNA knockdown, glucose-stimulated insulin secretion in INS-1 cells and human islets, and phospholipid profiling\",\n      \"pmids\": [\"28193492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between acyl-CoA synthesis and secretion not defined\", \"Whether granule targeting requires specific partners unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined ACSL3's distinct subcellular itinerary (trans-Golgi/endosomes and lipid droplets), separating it spatially from the ER-resident ACSL4.\",\n      \"evidence\": \"Subcellular fractionation, confocal immunofluorescence, and tumor array immunohistochemistry in fibrosarcoma and breast cancer cells\",\n      \"pmids\": [\"29450800\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Targeting determinants for Golgi/LD localization not mapped\", \"Functional consequence of compartment-specific localization not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed ACSL3 supplies arachidonate-derived phosphatidylinositols to an LPIAT1 axis sustaining prostaglandin synthesis, a tumor-promoting lipid signaling output.\",\n      \"evidence\": \"ACSL3/LPIAT1 knockdown in lung cancer lines and KrasG12D mice, lipidomics, prostaglandin measurement, and in vivo tumorigenesis assays\",\n      \"pmids\": [\"32034305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic flux of arachidonate through ACSL3 to PI not isotopically traced\", \"Generalizability beyond lung cancer not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected ACSL3 to autophagy machinery via direct binding to GABARAPL2 and ER recruitment of the UFM1 conjugation system.\",\n      \"evidence\": \"CRISPR endogenous ATG8 tagging, affinity purification-MS interactome, Co-IP, and knockdown of ufmylation/ER-phagy components\",\n      \"pmids\": [\"32843575\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ACSL3 LIR-mediated binding requires lipid catalysis unclear\", \"Physiological role of ACSL3-UFM1 link in vivo not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated ACSL3 dependence for lipid droplet accumulation from exogenous fatty acids and identified its role in shaping ferroptosis susceptibility in cancer.\",\n      \"evidence\": \"Genetic/pharmacologic ACSL3 inhibition, isotope-tracing lipidomics, orthotopic ccRCC mouse model, and FACS ferroptosis assays\",\n      \"pmids\": [\"36192773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lipid species mediating ferroptosis modulation not fully resolved\", \"Dependence on fatty acid composition mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established an ACSL3-PAI-1 axis by which ACSL3 reshapes the tumor immune microenvironment in pancreatic cancer.\",\n      \"evidence\": \"Acsl3 genetic KO in mouse PDAC, in vivo tumor growth, immune infiltrate flow cytometry, and PAI-1 measurement/inhibition with therapy response\",\n      \"pmids\": [\"33127675\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ACSL3 metabolic activity controls PAI-1 secretion is unclear\", \"Whether the effect is fully lipid-dependent untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed ACSL3 within a Rab18-PLIN2 lipid droplet complex regulating triacylglycerol levels and LD dynamics.\",\n      \"evidence\": \"Co-IP, Rab18 overexpression/knockdown, lipid droplet staining, and TAG quantification in C2C12 myoblasts\",\n      \"pmids\": [\"33713834\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single primary binding method without reciprocal structural validation\", \"Direct vs indirect nature of complex assembly unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved post-translational control of ACSL3 stability, showing E3 ligase TRIM25 (recruited by ANKRD1) drives K63-ubiquitination and degradation to amplify ferroptosis.\",\n      \"evidence\": \"IP-MS, reciprocal Co-IP, proximity ligation, K63-linkage ubiquitination assays, and in vivo rAAV9 knockdown in renal ischemia-reperfusion\",\n      \"pmids\": [\"39285846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K63-ubiquitination targets ACSL3 to proteasome or another fate unclear\", \"Other adaptors recruiting TRIM25 to ACSL3 unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the ACSL3 degradation network to NEDD4 (via TNFAIP3) and HRD1, defining multiple converging ubiquitin pathways that tune ACSL3 levels in disease.\",\n      \"evidence\": \"Co-IP, ubiquitination assays identifying E3 ligases, and in vivo AAV knockdown in TBI and ethanol-diet liver injury models\",\n      \"pmids\": [\"39743027\", \"41130543\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of each E3 ligase across tissues not compared\", \"Ubiquitination sites on ACSL3 not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified stabilizing factors SUMO2 and NT5DC2 that block ACSL3 ubiquitination, establishing a balance between degradation and protection that sets ferroptosis sensitivity.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, and ACSL3 rescue/knockdown ferroptosis experiments in hepatocellular and bladder cancer cells\",\n      \"pmids\": [\"40526170\", \"41974665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SUMO2 acts via direct SUMOylation of ACSL3 not resolved\", \"Single-lab Co-IP evidence per partner without structural validation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that ACSL3 enzymatic activity directly governs the MUFA/PUFA phospholipid ratio and can be pharmacologically enhanced (vitamin A/ATRA) to suppress ferroptosis and extend lifespan.\",\n      \"evidence\": \"In vitro enzymatic activity/binding assays, phospholipid lipidomics, ferroptosis assays, and ACSL3-dependent C. elegans lifespan analysis\",\n      \"pmids\": [\"41909752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of vitamin A binding to ACSL3 not solved\", \"Mammalian lifespan/healthspan effects not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Refined ACSL3's catalytic-activity-independent role in autophagy to an early step of fusion-competent autophagosomal membrane formation, regulated by SYNTAXIN17.\",\n      \"evidence\": \"Knockdown/overexpression with enzymatic-dead mutants, WIPI2/LC3/FIP200 imaging, LC3 lipidation assays, and SYNTAXIN17 epistasis\",\n      \"pmids\": [\"40728409\", \"41346954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which ACSL3 confers membrane fusion competence unknown\", \"How ACSL3 partitions between LD biogenesis and autophagosome roles unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Positioned ACSL3 as a central genome-wide determinant of hepatocyte palmitate lipotoxicity, integrating its substrate routing into a tractable therapeutic node.\",\n      \"evidence\": \"Genome-wide CRISPR screen with genetic/pharmacologic validation, isotope tracing of fatty acid incorporation, and human MASLD tissue with spatial transcriptomics\",\n      \"pmids\": [\"41564380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether catalytic vs non-catalytic functions drive lipotoxicity not dissected\", \"Long-term consequences of ACSL3 inhibition in liver not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ACSL3 mechanistically toggles between its catalytic functions (fatty acid activation, phospholipid remodeling, ferroptosis control) and its catalytic-activity-independent functions (Lyn export, autophagosome membrane formation) within a single protein remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model coupling catalytic and scaffolding activities\", \"Determinants directing ACSL3 to Golgi vs lipid droplet vs autophagosomal membranes unknown\", \"Ubiquitination/SUMOylation site mapping incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 2, 26]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 9, 26]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [8, 10, 13]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [11, 22]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 10, 32]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [10, 17, 26]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [11, 24, 25]}\n    ],\n    \"complexes\": [\"Rab18-PLIN2-ACSL3 lipid droplet complex\"],\n    \"partners\": [\"LYN\", \"GABARAPL2\", \"RAB18\", \"PLIN2\", \"YES1\", \"NT5DC2\", \"TRIM25\", \"ANKRD1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}