{"gene":"TSPAN9","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2009,"finding":"TSPAN9 is expressed on the platelet surface (~2800 copies per platelet) and is a component of tetraspanin-enriched microdomains that include the collagen receptor GPVI and integrin α6β1, but not GPIbα, αIIbβ3, or α2β1 integrins.","method":"Co-immunoprecipitation, antibody generation, flow cytometry, and serial analysis of gene expression/DNA microarrays in megakaryocytes and platelets","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with multiple associated proteins tested, single lab, multiple orthogonal methods (Co-IP, flow cytometry, SAGE/microarray)","pmids":["18795891"],"is_preprint":false},{"year":2016,"finding":"TSPAN9 co-immunoprecipitates and co-localizes with GPVI on platelets; genetic deletion of Tspan9 in mice causes defective GPVI-induced platelet aggregation, secretion, and protein tyrosine phosphorylation. Super-resolution imaging showed no defect in collagen-induced GPVI clustering, but single-particle tracking (TIRF) revealed ~50% reduction in GPVI lateral diffusion in the absence of Tspan9, indicating that Tspan9 promotes GPVI membrane dynamics rather than clustering.","method":"Gene-trap knockout mice, co-immunoprecipitation, co-localization imaging, super-resolution microscopy, single-particle tracking by TIRF microscopy, platelet aggregation and secretion assays, tyrosine phosphorylation western blot","journal":"Platelets","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genetic KO with defined cellular phenotype, reciprocal Co-IP, super-resolution imaging, and single-particle tracking providing mechanistic resolution; multiple orthogonal methods in one study","pmids":["28032533"],"is_preprint":false},{"year":2016,"finding":"TSPAN9 overexpression in gastric cancer SGC7901 cells inhibits proliferation, migration, and invasion by downregulating ERK1/2 phosphorylation and reducing secretion of MMP-9 and uPA.","method":"CCK-8 assay, cell cycle analysis, wound-healing assay, Transwell assay, western blot (pERK1/2), ELISA (MMP-9, uPA) with TSPAN9 overexpression","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss/gain-of-function with defined phenotypic readouts and pathway marker measurement; single lab, multiple functional assays but no direct pathway epistasis rescue","pmids":["27177197"],"is_preprint":false},{"year":2016,"finding":"TSPAN9 localizes to the plasma membrane and early/late endosomes; depletion of TSPAN9 inhibits membrane fusion of alphaviruses (SFV, SINV, CHIKV) and VSV in early endosomes without altering SFV delivery to early endosomes or changing their pH or protease activity, while late-endosome-fusing viruses are largely unaffected. TSPAN9 depletion also reduced levels of late endosomal proteins LAMP1 and CD63 and increased LAMP2.","method":"siRNA depletion in U-2 OS cells stably overexpressing TSPAN9, confocal colocalization, membrane fusion assays, viral infection assays with multiple viruses, endosomal pH/protease activity measurements, western blot for endosomal markers","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific functional readout (virus fusion), multiple virus comparisons providing mechanistic specificity; single lab","pmids":["26865714"],"is_preprint":false},{"year":2019,"finding":"TSPAN9 co-localizes and co-immunoprecipitates with EMILIN1 in gastric cancer cells; EMILIN1 overexpression upregulates TSPAN9 expression and synergistically enhances TSPAN9-mediated suppression of gastric cancer cell migration and invasion.","method":"Immunofluorescence co-localization, co-immunoprecipitation, western blot, wound-healing and invasion assays with TSPAN9 overexpression/knockdown and EMILIN1 overexpression","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus co-localization with functional assays; single lab, two orthogonal methods for interaction","pmids":["31242895"],"is_preprint":false},{"year":2020,"finding":"TSPAN9 interacts with PI3K and inhibits its catalytic activity, thereby downregulating the PI3K/AKT/mTOR pathway and promoting autophagy, which contributes to 5-FU resistance in gastric cancer cells.","method":"Co-immunoprecipitation, western blot for PI3K/AKT/mTOR pathway components, CCK-8 proliferation assay, gain/loss-of-function experiments in parental and 5-FU-resistant gastric cancer cells","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus biochemical pathway analysis with gain/loss-of-function; single lab, mechanistic claim of PI3K catalytic inhibition not directly reconstituted in vitro","pmids":["31911756"],"is_preprint":false},{"year":2022,"finding":"TSPAN9 interacts with β1 integrin in osteosarcoma cells (identified by mass spectrometry and confirmed by Co-IP) and promotes cell migration, invasion, and EMT via activation of the FAK/Ras/ERK1/2 signaling pathway; Tspan9 knockdown suppresses lung metastasis in a mouse tail-vein model.","method":"Mass spectrometry, co-immunoprecipitation, western blot, CCK-8, Transwell, wound-healing assays, in vivo tail-vein metastasis model in nude mice, siRNA knockdown and overexpression","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification of interacting partner confirmed by Co-IP, in vivo validation, pathway marker analysis; single lab","pmids":["35280793"],"is_preprint":false},{"year":2025,"finding":"TSPAN9 interacts with p62 (SQSTM1), impairs its cargo-receptor function, and thereby suppresses autophagy flux in cardiomyocytes; AAV9-mediated Tspan9 overexpression exacerbates TAC-induced cardiac hypertrophy and failure, while knockdown alleviates these phenotypes, and the protective effect of knockdown is abolished when autophagy is inactivated or p62 is knocked down.","method":"Co-immunoprecipitation (Tspan9–p62 interaction), AAV9-mediated cardiac overexpression and knockdown in mice with TAC model, neonatal rat cardiomyocyte PE-induced hypertrophy model, autophagy inhibition and p62 knockdown rescue epistasis","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus in vivo gain/loss-of-function with genetic epistasis (autophagy inhibition and p62 KD rescue), multiple orthogonal models (in vivo TAC + in vitro NRCMs), single lab but rigorous design","pmids":["40406987"],"is_preprint":false},{"year":2026,"finding":"TSPAN9 overexpression in IL-1β-induced senescent rat chondrocytes promotes mitocytosis (mitochondria extrusion), restores mitochondrial membrane potential, and reduces senescence markers and ECM catabolism; TSPAN9 acts in concert with KIF5B and TSPAN4 in this process.","method":"Lentiviral TSPAN9 overexpression in primary rat chondrocytes, SA-β-gal staining, JC-1 mitochondrial membrane potential assay, transmission electron microscopy, qRT-PCR for senescence/ECM/mitocytosis-related genes","journal":"Chinese journal of reparative and reconstructive surgery","confidence":"Low","confidence_rationale":"Tier 3 / Weak — gain-of-function with morphological and mRNA readouts; single lab, interaction with KIF5B/TSPAN4 not directly demonstrated by Co-IP or biochemical assay, abstract-level detail only","pmids":["41981440"],"is_preprint":false}],"current_model":"TSPAN9 is a tetraspanin membrane protein that organizes specialized microdomains: in platelets it associates with GPVI and promotes its lateral diffusion to facilitate GPVI-mediated activation; in early endosomes it modulates membrane fusion competence for early-penetrating viruses; in cardiomyocytes it interacts with p62/SQSTM1 to suppress autophagy and exacerbate hypertrophy; in cancer cells it engages β1 integrin and modulates FAK/Ras/ERK1/2 or PI3K/AKT/mTOR signaling to regulate migration, invasion, and drug resistance depending on cellular context."},"narrative":{"mechanistic_narrative":"TSPAN9 is a tetraspanin membrane protein that organizes specialized membrane microdomains and tunes signaling and membrane-trafficking events across diverse cell types [PMID:18795891, PMID:28032533]. In platelets, TSPAN9 is a component of tetraspanin-enriched microdomains containing the collagen receptor GPVI and integrin α6β1, and it associates directly with GPVI; genetic deletion in mice impairs GPVI-induced platelet aggregation, secretion, and tyrosine phosphorylation, acting not by altering GPVI clustering but by promoting GPVI lateral diffusion within the membrane [PMID:18795891, PMID:28032533]. TSPAN9 also localizes to the plasma membrane and endosomes, where it is required for the early-endosomal membrane fusion of certain enveloped viruses without affecting endosomal pH, protease activity, or cargo delivery, and its depletion shifts late-endosomal protein composition [PMID:26865714]. In disease contexts TSPAN9 modulates intracellular signaling and autophagy: in cardiomyocytes it binds p62/SQSTM1, impairs its cargo-receptor function, and suppresses autophagic flux, thereby exacerbating pressure-overload cardiac hypertrophy and failure in a manner dependent on autophagy and p62 [PMID:40406987]. In cancer cells TSPAN9 engages partners including β1 integrin and PI3K, and its effects on migration, invasion, and drug resistance are context-dependent—suppressing ERK1/2 signaling and EMILIN1-cooperative invasion in gastric cancer while activating FAK/Ras/ERK1/2 signaling to drive osteosarcoma metastasis [PMID:27177197, PMID:31242895, PMID:31911756, PMID:35280793].","teleology":[{"year":2009,"claim":"Established that TSPAN9 is a bona fide platelet surface tetraspanin and defined its selective microdomain partners, addressing whether it physically associates with specific platelet receptors.","evidence":"Co-immunoprecipitation, antibody/flow cytometry, and SAGE/microarray in megakaryocytes and platelets","pmids":["18795891"],"confidence":"Medium","gaps":["Functional consequence of the GPVI/α6β1 association not tested","Mechanism of selective partner inclusion/exclusion unresolved"]},{"year":2016,"claim":"Resolved how TSPAN9 supports GPVI function by showing it promotes receptor lateral diffusion rather than clustering, settling the mechanistic basis of its platelet role.","evidence":"Tspan9 gene-trap knockout mice, reciprocal Co-IP, super-resolution imaging, single-particle tracking by TIRF, aggregation/secretion and phosphorylation assays","pmids":["28032533"],"confidence":"High","gaps":["Structural basis of TSPAN9–GPVI interaction unknown","Whether TSPAN9 affects other platelet receptors in vivo untested"]},{"year":2016,"claim":"Identified a trafficking role for TSPAN9 distinct from its platelet function, showing it is required for early-endosomal viral membrane fusion and influences endosomal protein composition.","evidence":"siRNA depletion in U-2 OS cells, confocal colocalization, membrane fusion and multi-virus infection assays, endosomal pH/protease measurements, endosomal marker westerns","pmids":["26865714"],"confidence":"Medium","gaps":["Molecular mechanism by which TSPAN9 confers fusion competence unknown","Direct endosomal interaction partners not identified"]},{"year":2016,"claim":"Provided first evidence that TSPAN9 acts as a migration/invasion suppressor in gastric cancer via ERK1/2 pathway downregulation.","evidence":"TSPAN9 overexpression in SGC7901 cells with proliferation, wound-healing, Transwell assays, pERK1/2 western, MMP-9/uPA ELISA","pmids":["27177197"],"confidence":"Medium","gaps":["No pathway epistasis rescue","Mechanism linking TSPAN9 to ERK1/2 not defined"]},{"year":2019,"claim":"Linked TSPAN9 to EMILIN1 as a cooperating partner that amplifies its tumor-suppressive effect in gastric cancer.","evidence":"Immunofluorescence co-localization, Co-IP, western blot, wound-healing and invasion assays with TSPAN9 and EMILIN1 manipulation","pmids":["31242895"],"confidence":"Medium","gaps":["Direct vs indirect nature of TSPAN9–EMILIN1 interaction unresolved","Mechanism of reciprocal expression upregulation unknown"]},{"year":2020,"claim":"Connected TSPAN9 to PI3K/AKT/mTOR signaling and autophagy-mediated chemoresistance, proposing direct inhibition of PI3K catalytic activity.","evidence":"Co-IP, PI3K/AKT/mTOR pathway westerns, CCK-8, gain/loss-of-function in parental and 5-FU-resistant gastric cancer cells","pmids":["31911756"],"confidence":"Medium","gaps":["PI3K catalytic inhibition not reconstituted in vitro","Apparent contradiction with tumor-suppressive role across gastric cancer studies not reconciled"]},{"year":2022,"claim":"Demonstrated a pro-metastatic role for TSPAN9 in osteosarcoma via β1 integrin engagement and FAK/Ras/ERK1/2 activation, contrasting its gastric cancer phenotype.","evidence":"Mass spectrometry, Co-IP, pathway westerns, CCK-8/Transwell/wound-healing assays, in vivo tail-vein metastasis in nude mice with knockdown/overexpression","pmids":["35280793"],"confidence":"Medium","gaps":["Basis for opposite signaling outcomes across cancer types unexplained","Direct β1 integrin binding interface not mapped"]},{"year":2025,"claim":"Established a cardiac role through which TSPAN9 binds p62/SQSTM1, impairs cargo-receptor function, and suppresses autophagy to drive hypertrophy, with genetic epistasis confirming the pathway.","evidence":"Co-IP, AAV9 cardiac overexpression/knockdown in mouse TAC model, neonatal rat cardiomyocyte hypertrophy model, autophagy-inhibition and p62-knockdown rescue epistasis","pmids":["40406987"],"confidence":"High","gaps":["Molecular mechanism by which TSPAN9 impairs p62 cargo function unknown","Relationship to TSPAN9's PI3K-autophagy axis in cancer not addressed"]},{"year":2026,"claim":"Implicated TSPAN9 in mitochondrial quality control by promoting mitocytosis in senescent chondrocytes alongside KIF5B and TSPAN4.","evidence":"Lentiviral TSPAN9 overexpression in IL-1β-induced rat chondrocytes, SA-β-gal, JC-1, TEM, qRT-PCR","pmids":["41981440"],"confidence":"Low","gaps":["Interaction with KIF5B/TSPAN4 not demonstrated by Co-IP or biochemistry","Abstract-level detail only; mechanism of mitocytosis induction unknown"]},{"year":null,"claim":"How a single tetraspanin produces opposite signaling outcomes (suppressive vs activating ERK; autophagy-suppressing vs autophagy-promoting) across cell types remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying biochemical model reconciling context-dependent partner engagement","No structural data on TSPAN9 microdomain organization","Direct vs adaptor-mediated nature of most reported interactions undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,5,7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[5,7]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,5,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3]}],"complexes":["tetraspanin-enriched microdomain"],"partners":["GPVI","ITGB1","PIK3","SQSTM1","EMILIN1","ITGA6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75954","full_name":"Tetraspanin-9","aliases":["Tetraspan NET-5"],"length_aa":239,"mass_kda":26.8,"function":"","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/O75954/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TSPAN9","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TSPAN9","total_profiled":1310},"omim":[{"mim_id":"613137","title":"TETRASPANIN 9; TSPAN9","url":"https://www.omim.org/entry/613137"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":148.5},{"tissue":"heart muscle","ntpm":120.6}],"url":"https://www.proteinatlas.org/search/TSPAN9"},"hgnc":{"alias_symbol":["NET-5"],"prev_symbol":[]},"alphafold":{"accession":"O75954","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75954","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75954-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75954-F1-predicted_aligned_error_v6.png","plddt_mean":89.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TSPAN9","jax_strain_url":"https://www.jax.org/strain/search?query=TSPAN9"},"sequence":{"accession":"O75954","fasta_url":"https://rest.uniprot.org/uniprotkb/O75954.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75954/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75954"}},"corpus_meta":[{"pmid":"18795891","id":"PMC_18795891","title":"Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains.","date":"2009","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/18795891","citation_count":61,"is_preprint":false},{"pmid":"31242895","id":"PMC_31242895","title":"TSPAN9 and EMILIN1 synergistically inhibit the migration and invasion of gastric cancer cells by increasing TSPAN9 expression.","date":"2019","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31242895","citation_count":33,"is_preprint":false},{"pmid":"31911756","id":"PMC_31911756","title":"TSPAN9 suppresses the chemosensitivity of gastric cancer to 5-fluorouracil by promoting autophagy.","date":"2020","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/31911756","citation_count":27,"is_preprint":false},{"pmid":"27177197","id":"PMC_27177197","title":"Tspan9 inhibits the proliferation, migration and invasion of human gastric cancer SGC7901 cells via the ERK1/2 pathway.","date":"2016","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/27177197","citation_count":25,"is_preprint":false},{"pmid":"28032533","id":"PMC_28032533","title":"Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation.","date":"2016","source":"Platelets","url":"https://pubmed.ncbi.nlm.nih.gov/28032533","citation_count":23,"is_preprint":false},{"pmid":"35280793","id":"PMC_35280793","title":"Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35280793","citation_count":21,"is_preprint":false},{"pmid":"26865714","id":"PMC_26865714","title":"Role of TSPAN9 in Alphavirus Entry and Early Endosomes.","date":"2016","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/26865714","citation_count":20,"is_preprint":false},{"pmid":"40406987","id":"PMC_40406987","title":"Tspan9 Exacerbates Cardiac Hypertrophy by Impairing Cardiac Autophagy.","date":"2025","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/40406987","citation_count":0,"is_preprint":false},{"pmid":"40792132","id":"PMC_40792132","title":"TSPAN9 inhibits malignant progression of hepatocellular carcinoma.","date":"2025","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/40792132","citation_count":0,"is_preprint":false},{"pmid":"41981440","id":"PMC_41981440","title":"[Protective effect and mechanism of TSPAN9-mediated mitocytosis in interleukin-1β-induced rat chondrocyte senescence].","date":"2026","source":"Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery","url":"https://pubmed.ncbi.nlm.nih.gov/41981440","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.19.629410","title":"Genome-wide association analyses in dairy heifers highlight genes overlapping with mouse and human fertility and human health traits","date":"2024-12-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.19.629410","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9148,"output_tokens":2600,"usd":0.033222,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9865,"output_tokens":3426,"usd":0.067488,"stage2_stop_reason":"end_turn"},"total_usd":0.10071,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"TSPAN9 is expressed on the platelet surface (~2800 copies per platelet) and is a component of tetraspanin-enriched microdomains that include the collagen receptor GPVI and integrin α6β1, but not GPIbα, αIIbβ3, or α2β1 integrins.\",\n      \"method\": \"Co-immunoprecipitation, antibody generation, flow cytometry, and serial analysis of gene expression/DNA microarrays in megakaryocytes and platelets\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with multiple associated proteins tested, single lab, multiple orthogonal methods (Co-IP, flow cytometry, SAGE/microarray)\",\n      \"pmids\": [\"18795891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TSPAN9 co-immunoprecipitates and co-localizes with GPVI on platelets; genetic deletion of Tspan9 in mice causes defective GPVI-induced platelet aggregation, secretion, and protein tyrosine phosphorylation. Super-resolution imaging showed no defect in collagen-induced GPVI clustering, but single-particle tracking (TIRF) revealed ~50% reduction in GPVI lateral diffusion in the absence of Tspan9, indicating that Tspan9 promotes GPVI membrane dynamics rather than clustering.\",\n      \"method\": \"Gene-trap knockout mice, co-immunoprecipitation, co-localization imaging, super-resolution microscopy, single-particle tracking by TIRF microscopy, platelet aggregation and secretion assays, tyrosine phosphorylation western blot\",\n      \"journal\": \"Platelets\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genetic KO with defined cellular phenotype, reciprocal Co-IP, super-resolution imaging, and single-particle tracking providing mechanistic resolution; multiple orthogonal methods in one study\",\n      \"pmids\": [\"28032533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TSPAN9 overexpression in gastric cancer SGC7901 cells inhibits proliferation, migration, and invasion by downregulating ERK1/2 phosphorylation and reducing secretion of MMP-9 and uPA.\",\n      \"method\": \"CCK-8 assay, cell cycle analysis, wound-healing assay, Transwell assay, western blot (pERK1/2), ELISA (MMP-9, uPA) with TSPAN9 overexpression\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss/gain-of-function with defined phenotypic readouts and pathway marker measurement; single lab, multiple functional assays but no direct pathway epistasis rescue\",\n      \"pmids\": [\"27177197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TSPAN9 localizes to the plasma membrane and early/late endosomes; depletion of TSPAN9 inhibits membrane fusion of alphaviruses (SFV, SINV, CHIKV) and VSV in early endosomes without altering SFV delivery to early endosomes or changing their pH or protease activity, while late-endosome-fusing viruses are largely unaffected. TSPAN9 depletion also reduced levels of late endosomal proteins LAMP1 and CD63 and increased LAMP2.\",\n      \"method\": \"siRNA depletion in U-2 OS cells stably overexpressing TSPAN9, confocal colocalization, membrane fusion assays, viral infection assays with multiple viruses, endosomal pH/protease activity measurements, western blot for endosomal markers\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific functional readout (virus fusion), multiple virus comparisons providing mechanistic specificity; single lab\",\n      \"pmids\": [\"26865714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TSPAN9 co-localizes and co-immunoprecipitates with EMILIN1 in gastric cancer cells; EMILIN1 overexpression upregulates TSPAN9 expression and synergistically enhances TSPAN9-mediated suppression of gastric cancer cell migration and invasion.\",\n      \"method\": \"Immunofluorescence co-localization, co-immunoprecipitation, western blot, wound-healing and invasion assays with TSPAN9 overexpression/knockdown and EMILIN1 overexpression\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus co-localization with functional assays; single lab, two orthogonal methods for interaction\",\n      \"pmids\": [\"31242895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TSPAN9 interacts with PI3K and inhibits its catalytic activity, thereby downregulating the PI3K/AKT/mTOR pathway and promoting autophagy, which contributes to 5-FU resistance in gastric cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, western blot for PI3K/AKT/mTOR pathway components, CCK-8 proliferation assay, gain/loss-of-function experiments in parental and 5-FU-resistant gastric cancer cells\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus biochemical pathway analysis with gain/loss-of-function; single lab, mechanistic claim of PI3K catalytic inhibition not directly reconstituted in vitro\",\n      \"pmids\": [\"31911756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TSPAN9 interacts with β1 integrin in osteosarcoma cells (identified by mass spectrometry and confirmed by Co-IP) and promotes cell migration, invasion, and EMT via activation of the FAK/Ras/ERK1/2 signaling pathway; Tspan9 knockdown suppresses lung metastasis in a mouse tail-vein model.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, western blot, CCK-8, Transwell, wound-healing assays, in vivo tail-vein metastasis model in nude mice, siRNA knockdown and overexpression\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification of interacting partner confirmed by Co-IP, in vivo validation, pathway marker analysis; single lab\",\n      \"pmids\": [\"35280793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TSPAN9 interacts with p62 (SQSTM1), impairs its cargo-receptor function, and thereby suppresses autophagy flux in cardiomyocytes; AAV9-mediated Tspan9 overexpression exacerbates TAC-induced cardiac hypertrophy and failure, while knockdown alleviates these phenotypes, and the protective effect of knockdown is abolished when autophagy is inactivated or p62 is knocked down.\",\n      \"method\": \"Co-immunoprecipitation (Tspan9–p62 interaction), AAV9-mediated cardiac overexpression and knockdown in mice with TAC model, neonatal rat cardiomyocyte PE-induced hypertrophy model, autophagy inhibition and p62 knockdown rescue epistasis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus in vivo gain/loss-of-function with genetic epistasis (autophagy inhibition and p62 KD rescue), multiple orthogonal models (in vivo TAC + in vitro NRCMs), single lab but rigorous design\",\n      \"pmids\": [\"40406987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TSPAN9 overexpression in IL-1β-induced senescent rat chondrocytes promotes mitocytosis (mitochondria extrusion), restores mitochondrial membrane potential, and reduces senescence markers and ECM catabolism; TSPAN9 acts in concert with KIF5B and TSPAN4 in this process.\",\n      \"method\": \"Lentiviral TSPAN9 overexpression in primary rat chondrocytes, SA-β-gal staining, JC-1 mitochondrial membrane potential assay, transmission electron microscopy, qRT-PCR for senescence/ECM/mitocytosis-related genes\",\n      \"journal\": \"Chinese journal of reparative and reconstructive surgery\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — gain-of-function with morphological and mRNA readouts; single lab, interaction with KIF5B/TSPAN4 not directly demonstrated by Co-IP or biochemical assay, abstract-level detail only\",\n      \"pmids\": [\"41981440\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TSPAN9 is a tetraspanin membrane protein that organizes specialized microdomains: in platelets it associates with GPVI and promotes its lateral diffusion to facilitate GPVI-mediated activation; in early endosomes it modulates membrane fusion competence for early-penetrating viruses; in cardiomyocytes it interacts with p62/SQSTM1 to suppress autophagy and exacerbate hypertrophy; in cancer cells it engages β1 integrin and modulates FAK/Ras/ERK1/2 or PI3K/AKT/mTOR signaling to regulate migration, invasion, and drug resistance depending on cellular context.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TSPAN9 is a tetraspanin membrane protein that organizes specialized membrane microdomains and tunes signaling and membrane-trafficking events across diverse cell types [#0, #1]. In platelets, TSPAN9 is a component of tetraspanin-enriched microdomains containing the collagen receptor GPVI and integrin α6β1, and it associates directly with GPVI; genetic deletion in mice impairs GPVI-induced platelet aggregation, secretion, and tyrosine phosphorylation, acting not by altering GPVI clustering but by promoting GPVI lateral diffusion within the membrane [#0, #1]. TSPAN9 also localizes to the plasma membrane and endosomes, where it is required for the early-endosomal membrane fusion of certain enveloped viruses without affecting endosomal pH, protease activity, or cargo delivery, and its depletion shifts late-endosomal protein composition [#3]. In disease contexts TSPAN9 modulates intracellular signaling and autophagy: in cardiomyocytes it binds p62/SQSTM1, impairs its cargo-receptor function, and suppresses autophagic flux, thereby exacerbating pressure-overload cardiac hypertrophy and failure in a manner dependent on autophagy and p62 [#7]. In cancer cells TSPAN9 engages partners including β1 integrin and PI3K, and its effects on migration, invasion, and drug resistance are context-dependent—suppressing ERK1/2 signaling and EMILIN1-cooperative invasion in gastric cancer while activating FAK/Ras/ERK1/2 signaling to drive osteosarcoma metastasis [#2, #4, #5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that TSPAN9 is a bona fide platelet surface tetraspanin and defined its selective microdomain partners, addressing whether it physically associates with specific platelet receptors.\",\n      \"evidence\": \"Co-immunoprecipitation, antibody/flow cytometry, and SAGE/microarray in megakaryocytes and platelets\",\n      \"pmids\": [\"18795891\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the GPVI/α6β1 association not tested\", \"Mechanism of selective partner inclusion/exclusion unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved how TSPAN9 supports GPVI function by showing it promotes receptor lateral diffusion rather than clustering, settling the mechanistic basis of its platelet role.\",\n      \"evidence\": \"Tspan9 gene-trap knockout mice, reciprocal Co-IP, super-resolution imaging, single-particle tracking by TIRF, aggregation/secretion and phosphorylation assays\",\n      \"pmids\": [\"28032533\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TSPAN9–GPVI interaction unknown\", \"Whether TSPAN9 affects other platelet receptors in vivo untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a trafficking role for TSPAN9 distinct from its platelet function, showing it is required for early-endosomal viral membrane fusion and influences endosomal protein composition.\",\n      \"evidence\": \"siRNA depletion in U-2 OS cells, confocal colocalization, membrane fusion and multi-virus infection assays, endosomal pH/protease measurements, endosomal marker westerns\",\n      \"pmids\": [\"26865714\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism by which TSPAN9 confers fusion competence unknown\", \"Direct endosomal interaction partners not identified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided first evidence that TSPAN9 acts as a migration/invasion suppressor in gastric cancer via ERK1/2 pathway downregulation.\",\n      \"evidence\": \"TSPAN9 overexpression in SGC7901 cells with proliferation, wound-healing, Transwell assays, pERK1/2 western, MMP-9/uPA ELISA\",\n      \"pmids\": [\"27177197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No pathway epistasis rescue\", \"Mechanism linking TSPAN9 to ERK1/2 not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked TSPAN9 to EMILIN1 as a cooperating partner that amplifies its tumor-suppressive effect in gastric cancer.\",\n      \"evidence\": \"Immunofluorescence co-localization, Co-IP, western blot, wound-healing and invasion assays with TSPAN9 and EMILIN1 manipulation\",\n      \"pmids\": [\"31242895\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of TSPAN9–EMILIN1 interaction unresolved\", \"Mechanism of reciprocal expression upregulation unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected TSPAN9 to PI3K/AKT/mTOR signaling and autophagy-mediated chemoresistance, proposing direct inhibition of PI3K catalytic activity.\",\n      \"evidence\": \"Co-IP, PI3K/AKT/mTOR pathway westerns, CCK-8, gain/loss-of-function in parental and 5-FU-resistant gastric cancer cells\",\n      \"pmids\": [\"31911756\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PI3K catalytic inhibition not reconstituted in vitro\", \"Apparent contradiction with tumor-suppressive role across gastric cancer studies not reconciled\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated a pro-metastatic role for TSPAN9 in osteosarcoma via β1 integrin engagement and FAK/Ras/ERK1/2 activation, contrasting its gastric cancer phenotype.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, pathway westerns, CCK-8/Transwell/wound-healing assays, in vivo tail-vein metastasis in nude mice with knockdown/overexpression\",\n      \"pmids\": [\"35280793\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Basis for opposite signaling outcomes across cancer types unexplained\", \"Direct β1 integrin binding interface not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a cardiac role through which TSPAN9 binds p62/SQSTM1, impairs cargo-receptor function, and suppresses autophagy to drive hypertrophy, with genetic epistasis confirming the pathway.\",\n      \"evidence\": \"Co-IP, AAV9 cardiac overexpression/knockdown in mouse TAC model, neonatal rat cardiomyocyte hypertrophy model, autophagy-inhibition and p62-knockdown rescue epistasis\",\n      \"pmids\": [\"40406987\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which TSPAN9 impairs p62 cargo function unknown\", \"Relationship to TSPAN9's PI3K-autophagy axis in cancer not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Implicated TSPAN9 in mitochondrial quality control by promoting mitocytosis in senescent chondrocytes alongside KIF5B and TSPAN4.\",\n      \"evidence\": \"Lentiviral TSPAN9 overexpression in IL-1β-induced rat chondrocytes, SA-β-gal, JC-1, TEM, qRT-PCR\",\n      \"pmids\": [\"41981440\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Interaction with KIF5B/TSPAN4 not demonstrated by Co-IP or biochemistry\", \"Abstract-level detail only; mechanism of mitocytosis induction unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single tetraspanin produces opposite signaling outcomes (suppressive vs activating ERK; autophagy-suppressing vs autophagy-promoting) across cell types remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying biochemical model reconciling context-dependent partner engagement\", \"No structural data on TSPAN9 microdomain organization\", \"Direct vs adaptor-mediated nature of most reported interactions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 5, 7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [5, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 5, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\"tetraspanin-enriched microdomain\"],\n    \"partners\": [\"GPVI\", \"ITGB1\", \"PIK3\", \"SQSTM1\", \"EMILIN1\", \"ITGA6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}