{"gene":"SPAG6","run_date":"2026-06-10T07:46:38","timeline":{"discoveries":[{"year":1996,"finding":"PF16 (SPAG6 orthologue in Chlamydomonas) contains eight contiguous armadillo repeats and localizes specifically to the C1 microtubule of the central apparatus in flagella; loss of PF16 results in absence of the C1 microtubule and flagellar paralysis, and rescue by transformation restores wild-type motility and ultrastructure.","method":"Insertional mutagenesis, cDNA cloning, transformation rescue, immunofluorescence and immunogold electron microscopy with anti-PF16 antibody","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genetic rescue with defined phenotypic readout, direct localization by immunogold EM, replicated by multiple orthogonal methods in a single rigorous study","pmids":["8636214"],"is_preprint":false},{"year":2000,"finding":"Functional domain analysis of PF16 demonstrated that the first armadillo repeat is necessary but not sufficient for assembly into the axoneme; the C-terminal 122 amino acids are dispensable for assembly and motility; and the armadillo repeats function as a single structural unit required for PF16 assembly.","method":"Site-directed mutagenesis of PF16 locus alleles, deletion constructs transformed into pf16 mutants, motility rescue and ultrastructural analysis","journal":"Cell motility and the cytoskeleton","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vivo mutagenesis with functional rescue readout, multiple deletion constructs tested systematically in a single study","pmids":["10913963"],"is_preprint":false},{"year":1999,"finding":"Human SPAG6 protein, containing eight contiguous armadillo repeats homologous to Chlamydomonas PF16, localizes to the tails of permeabilized human sperm, consistent with a role in the axoneme central apparatus.","method":"cDNA cloning, immunofluorescence with anti-SPAG6 antibody on permeabilized human sperm","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment; single lab but corroborated by prior Chlamydomonas work and subsequent mammalian studies","pmids":["10493827"],"is_preprint":false},{"year":2000,"finding":"Mouse Spag6 protein (murine SPAG6) localizes to the principal piece of permeabilized sperm tails and co-localizes with microtubules when expressed in COS-1 cells, establishing it as a microtubule-associated axonemal central apparatus protein.","method":"Antipeptide antibody immunofluorescence on mouse sperm and COS-1 cells transfected with Spag6 expression construct","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by immunofluorescence in two cell systems, single lab","pmids":["10684790"],"is_preprint":false},{"year":2002,"finding":"Mammalian PF20 (SPAG16) interacts with SPAG6 via its WD repeat domain, as shown by yeast two-hybrid assay; when co-expressed in CHO cells, PF20 co-localizes with SPAG6 on polymerized microtubules; and PF20 protein is markedly reduced in sperm of Spag6-deficient mice, indicating SPAG6 is required for PF20 stability or assembly in the axoneme.","method":"Yeast two-hybrid, co-expression in CHO cells with fluorescence microscopy, immunoblotting of Spag6-knockout mouse sperm","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal evidence from yeast two-hybrid, co-localization, and in vivo knockout, multiple orthogonal methods across two proteins","pmids":["12391165"],"is_preprint":false},{"year":2005,"finding":"Mammalian PF6 (the orthologue of Chlamydomonas PF6, localized to the C1 1a projection) interacts with SPAG6 via a defined fragment, demonstrated by yeast two-hybrid and co-localization in transfected cells; a PF6 fragment corresponding to the SPAG6-binding domain is absent from sperm of SPAG6-deficient mice, establishing PF6–SPAG6–PF20 as a network linking C1 microtubule projections to the central bridge in the axoneme.","method":"Yeast two-hybrid, co-transfection and co-localization in mammalian cells, immunoblotting of Spag6-knockout mouse sperm","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (two-hybrid, co-localization, in vivo knockout), single lab but rigorous","pmids":["15827353"],"is_preprint":false},{"year":2014,"finding":"In Spag6-deficient mice, ependymal and tracheal cilia show reduced beat frequency, unsynchronized beating, reduced cilia density, and randomized basal foot orientation; planar cell polarity protein Vangl2 distribution is lost in tracheal epithelial cells, demonstrating that SPAG6 regulates not only ciliary motility but also ciliogenesis, axoneme orientation, and epithelial planar cell polarity.","method":"Spag6 knockout mouse analysis: high-speed video microscopy of cilia beat, scanning/transmission electron microscopy, immunofluorescence for basal feet orientation and Vangl2 distribution","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with multiple defined cellular phenotypes, orthogonal methods (video microscopy, EM, IF), single lab","pmids":["25333478"],"is_preprint":false},{"year":2014,"finding":"SPAG6 deficiency in mice leads to impaired immunological synapse formation due to loss of centrosome polarization and failure of actin clearance at the synaptic cleft in lymphocytes, resulting in defective CTL function and reduced humoral immunity (germinal centers, class-switched antibody); SPAG6 is associated with the centrosome in lymphocytes.","method":"Bone marrow reconstitution, immunofluorescence for centrosome polarization and actin distribution in lymphocytes, CTL killing assays, antibody production assays in Spag6-knockout mice","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with specific mechanistic phenotype (centrosome polarization), multiple immune functional readouts, single lab","pmids":["27169488"],"is_preprint":false},{"year":2014,"finding":"SPAG6 silencing in myeloid leukemia cells (SKM-1 and K562) induces apoptosis via activation of caspase-3, -9, and -8, and upregulates p53 and PTEN expression at mRNA and protein levels; SPAG6-shRNA xenograft mice showed inhibited tumor growth and increased apoptosis.","method":"shRNA lentiviral knockdown, flow cytometry, CCK-8 assay, Western blot, RT-PCR, NOD/SCID xenograft model","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with multiple readouts (apoptosis, caspase activation, p53/PTEN upregulation) in vitro and in vivo, single lab","pmids":["25405588"],"is_preprint":false},{"year":2015,"finding":"Spag6-deficient mouse embryonic fibroblasts proliferate more slowly, have larger surface area, reduced motility (chemotaxis and wound healing), reduced adhesion with non-polarized F-actin distribution, multiple centrosomes, reduced primary cilia formation, and increased sensitivity to paclitaxel; re-expression of SPAG6 rescues abnormal cell morphology; SPAG6 selectively increases acetylated tubulin levels.","method":"MEF isolation from Spag6-knockout embryos, proliferation assays, wound healing/chemotaxis assays, immunofluorescence for F-actin and tubulin, primary cilia quantification, rescue by SPAG6 re-expression","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with rescue, multiple orthogonal cellular assays, defined molecular correlate (acetylated tubulin), single lab","pmids":["26585507"],"is_preprint":false},{"year":2015,"finding":"Overexpression of Spag6 in cortical neurons delays neuronal migration rate and decreases neurite number and length, as assayed by in utero electroporation, identifying a role for SPAG6 in regulating neuronal migration through cytoskeletal remodeling.","method":"In utero electroporation of mouse embryonic cortex, immunofluorescence and quantitative analysis of neuronal position and neurite morphology","journal":"Journal of molecular neuroscience : MN","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain-of-function with specific phenotypic readout in vivo, single lab, single method","pmids":["26130477"],"is_preprint":false},{"year":2015,"finding":"SPAG6 co-immunoprecipitates with prestin in outer hair cells (OHCs); Spag6-knockout mice show reduced prestin expression at both protein and mRNA levels and abnormal OHC morphology, indicating SPAG6 is required for prestin stability and normal OHC mechanosensory function.","method":"Co-immunoprecipitation, Western blot, real-time PCR, immunofluorescence in Spag6-knockout mice","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP demonstrates interaction, KO shows functional consequence, single lab","pmids":["25748314"],"is_preprint":false},{"year":2017,"finding":"SPAG6 regulates apoptosis in SKM-1 MDS cells via the TRAIL signaling pathway: SPAG6 knockdown activates the TRAIL pathway and increases interaction between FADD and TRAIL death receptors, while high SPAG6 expression suppresses TRAIL signaling; SPAG6 does not affect expression of TRAIL death receptors except FADD.","method":"shRNA lentiviral knockdown, flow cytometry, CCK-8 assay, Western blot, co-immunoprecipitation for FADD–death receptor interaction","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with pathway-specific mechanistic readouts and co-IP, single lab","pmids":["28393201"],"is_preprint":false},{"year":2018,"finding":"SPAG6 silencing in SKM-1 cells increases PTEN expression and reduces AKT phosphorylation, leading to apoptosis via Mcl-1 downregulation, cytochrome c release, and increased caspase-9; the PI3K inhibitor LY294002 synergistically enhances this apoptosis; SPAG6-regulated PTEN expression is caspase-independent; SPAG6 knockdown is associated with DNMT1 downregulation, suggesting SPAG6 may control PTEN expression via DNA methylation.","method":"shRNA lentiviral knockdown, Western blot, flow cytometry, PI3K inhibitor treatment, pan-caspase inhibitor treatment, in vivo xenograft model","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with pharmacological validation of pathway, multiple readouts, single lab","pmids":["29749435"],"is_preprint":false},{"year":2019,"finding":"SPAG6 knockdown in SKM-1 cells inhibits cell proliferation by mediating G1-to-S cell cycle arrest, upregulating p27Kip1, and regulating the AKT/FOXO signaling pathway.","method":"shRNA knockdown, CCK-8, FACS cell cycle analysis, RT-qPCR, Western blot","journal":"DNA and cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with defined cell cycle and pathway readout, single lab","pmids":["30835546"],"is_preprint":false},{"year":2019,"finding":"SPAG6 interacts with SPINK2 (serine protease inhibitor Kazal-type 2) in the acrosome of round spermatids; SPAG6 is expressed in the acrosome during spermatogenesis (postnatal days 16–28); in SPAG6-knockout mice, SPINK2 expression and acrosomal localization are absent, indicating SPAG6 stabilizes SPINK2 during acrosome formation.","method":"Yeast two-hybrid, co-localization in CHO cells by immunofluorescence, Western blot and immunofluorescence in Spag6-knockout mice","journal":"Zhonghua nan ke xue = National journal of andrology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid plus co-localization plus in vivo KO, single lab","pmids":["32216237"],"is_preprint":false},{"year":2020,"finding":"SPAG6 silencing in SKM-1 cells induces autophagic cell death via the AMPK/mTOR/ULK1 pathway: knockdown upregulates p-AMPK and p-ULK1 and downregulates p-mTOR; autophagy inhibition with chloroquine or 3-methyladenine decreases SPAG6-knockdown-mediated apoptosis; AMPK inhibition (Compound C) attenuates both autophagy and apoptosis.","method":"shRNA lentiviral knockdown, Western blot, flow cytometry, autophagy inhibitor and AMPK inhibitor pharmacological experiments","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with pharmacological pathway validation, single lab","pmids":["32537026"],"is_preprint":false},{"year":2020,"finding":"SPAG6 promotes proliferation and inhibits apoptosis in Burkitt lymphoma cells via the PTEN/PI3K/AKT pathway: SPAG6 knockdown increases PTEN and reduces p-AKT, while PTEN siRNA or PTEN inhibitor SF1670 reverses the anti-proliferative and pro-apoptotic effects of SPAG6 depletion both in vitro and in vivo.","method":"shRNA/siRNA knockdown and overexpression, Western blot, flow cytometry, PTEN inhibitor treatment, in vivo xenograft","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain- and loss-of-function with pharmacological rescue of PTEN, in vitro and in vivo, single lab","pmids":["33000212"],"is_preprint":false},{"year":2022,"finding":"SPAG6 interacts with MYO1D (myosin 1D) as identified by co-immunoprecipitation and mass spectrometry; SPAG6 overexpression promotes translocation of MYO1D from cytosol to cell membrane, upregulating EGFR family expression and thereby activating PI3K/AKT and ERK signaling to promote AML cell proliferation and migration.","method":"Immunoprecipitation, mass spectrometry, Western blot for MYO1D localization (membrane vs. cytosol fractionation), in vitro proliferation/migration assays, in vivo xenograft","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS for interaction, subcellular fractionation for localization change, in vitro and in vivo functional validation, single lab","pmids":["35667090"],"is_preprint":false},{"year":2023,"finding":"SPAG6 knockdown in MPN cell lines promotes apoptosis, reduces G1-to-S cell cycle progression, and is associated with downregulated STAT1; forced SPAG6 expression promotes clone formation and G1-to-S progression; chromatin immunoprecipitation shows STAT1 binds the SPAG6 promoter, and dual-luciferase assay shows STAT1 promotes SPAG6 transcription, establishing a positive feedback between SPAG6 and STAT1.","method":"shRNA knockdown, overexpression, FACS cell cycle analysis, ChIP assay, dual-luciferase reporter assay, Western blot","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase assays establish STAT1–SPAG6 transcriptional relationship; functional KD/OE with defined pathway, single lab","pmids":["37681349"],"is_preprint":false},{"year":2023,"finding":"SPAG6 knockdown in B-ALL cell lines inhibits proliferation and promotes apoptosis via the TGF-β/Smad signaling pathway.","method":"Lentiviral knockdown, cell proliferation assays, flow cytometry for apoptosis, Western blot for TGF-β/Smad pathway components","journal":"International journal of hematology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — loss-of-function with pathway correlation but limited mechanistic validation, single lab, single method type","pmids":["38147275"],"is_preprint":false},{"year":2016,"finding":"TAC1 protein interacts with SPAG6 as shown by yeast two-hybrid; when co-transfected with SPAG6 in CHO cells, TAC1 localizes to microtubules rather than remaining diffusely cytoplasmic, indicating SPAG6 directs TAC1 to microtubules.","method":"Yeast two-hybrid, co-transfection and immunofluorescence co-localization in CHO and COS-1 cells, Western blot","journal":"Zhonghua nan ke xue = National journal of andrology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, yeast two-hybrid plus co-localization only, limited functional follow-up","pmids":["29282908"],"is_preprint":false},{"year":2022,"finding":"DNMT3b-mediated hypermethylation of the SPAG6 promoter leads to SPAG6 downregulation in lung squamous cell carcinoma; restored SPAG6 expression inhibits STAT1 and STAT3 transcription, reversing JAK/STAT pathway activation and suppressing malignant cell phenotype.","method":"Bisulfite sequencing, DNMT3b functional assays, dual-luciferase assay for STAT1/STAT3 transcription, CCK-8/EdU/Transwell assays","journal":"American journal of translational research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — methylation writer identified (DNMT3b), luciferase assay for downstream transcriptional effect, single lab","pmids":["36398260"],"is_preprint":false},{"year":2024,"finding":"SPAG6 overexpression in AML cells reduces ROS and malondialdehyde levels and promotes GSTP1 expression; elevated GSTP1 forms a complex with JNK, reducing p-JNK and inhibiting JNK pathway activation, thereby attenuating the pro-apoptotic effects of daunorubicin; SPAG6 knockdown diminishes mitochondrial membrane potential and increases apoptosis.","method":"TMT proteomics after SPAG6 knockdown, CCK-8, flow cytometry for ROS and apoptosis, Western blot, in vivo xenograft, IHC","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — proteomics plus functional validation of ROS/GSTP1/JNK axis, in vitro and in vivo, single lab","pmids":["39508041"],"is_preprint":false},{"year":2025,"finding":"SPAG6 directly interacts with DUSP1 (dual-specificity phosphatase 1) in multiple myeloma cells, and SPAG6 modulates downstream MAPK/ERK pathway activity by regulating DUSP1 activity; SPAG6 overexpression promotes proliferation, migration, and apoptosis resistance, while knockdown has opposite effects.","method":"Transcriptome sequencing, co-immunoprecipitation (implied from 'directly interacts'), RNA interference, overexpression, cell proliferation/apoptosis/migration assays","journal":"Frontiers in pharmacology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — interaction and pathway modulation reported but mechanistic detail of DUSP1 regulation is limited in abstract, single lab","pmids":["40535772"],"is_preprint":false},{"year":2025,"finding":"Both SPAG6 and SPAG6L bind SPINK2, but SPAG6 has approximately 10-fold higher binding affinity than SPAG6L; SPAG6 modulates testicular AKAP4 and SPAG16L levels; compound Spag6−/−;Spag6l+/− males are infertile with defective spermiogenesis (abnormal chromatin condensation, malformed acrosome and manchette, disorganized mitochondrial sheath and fibrous sheath); SPAG6 suppresses tubulin acetylation whereas SPAG6L enhances it, indicating antagonistic roles in microtubule regulation.","method":"Compound knockout mouse genetics, histological and ultrastructural analysis, binding affinity assays for SPINK2, Western blot for AKAP4 and SPAG16L, acetylated tubulin immunoblotting","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — compound KO genetics with multiple ultrastructural and molecular phenotypes, binding affinity comparison, preprint not yet peer-reviewed","pmids":["40777416"],"is_preprint":true},{"year":2023,"finding":"The SPAG6 c.900 T>C variant in exon 7 generates a new splice acceptor site that inhibits exon 7 skipping, thereby promoting Sertoli cell growth and maintaining normal blood-testis barrier function; the variant is associated with semen quality traits in pigs.","method":"RNA-seq analysis, Sanger sequencing, splice site reporter assay, Sertoli cell culture assays for growth and blood-testis barrier function","journal":"Animal genetics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — splice site creation mechanistically validated in cell culture with functional blood-testis barrier readout, single lab","pmids":["37211688"],"is_preprint":false},{"year":2025,"finding":"CryoEM of the Trypanosoma brucei flagellum central apparatus identified PF16 (SPAG6 orthologue) as a structural scaffold critical for central apparatus assembly and axonemal asymmetry; PF16 is stably associated with the C1/C2 microtubules.","method":"Cryogenic electron microscopy (cryoEM) structural analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — high-quality structural method (cryoEM), but in a non-mammalian organism (T. brucei) and preprint, not directly validated by mutagenesis","pmids":[],"is_preprint":true}],"current_model":"SPAG6 is an armadillo-repeat-containing protein that functions as a structural scaffold on the C1 microtubule of the axoneme central apparatus, where it stabilizes the C1 microtubule and interacts with PF20 (SPAG16), PF6, SPINK2, MYO1D, prestin, and TAC1 to maintain axoneme integrity and regulate flagellar/ciliary motility; beyond its canonical axonemal role, SPAG6 associates with cytoplasmic microtubules and the centrosome to regulate ciliogenesis, planar cell polarity, epithelial cell polarity, cell proliferation, migration, and immunological synapse formation, and in hematological malignancies it modulates apoptosis via TRAIL, PTEN/PI3K/AKT, AMPK/mTOR/ULK1, AKT/FOXO, and ROS/JNK/GSTP1 signaling pathways."},"narrative":{"mechanistic_narrative":"SPAG6 is an armadillo-repeat protein that functions as a structural scaffold of the central apparatus of motile cilia and flagella, where it stabilizes the C1 microtubule and organizes the projections that confer axonemal asymmetry [PMID:8636214]. First defined through the Chlamydomonas orthologue PF16, SPAG6 localizes specifically to the C1 microtubule, and its loss eliminates the C1 microtubule and paralyzes the flagellum, a phenotype rescued by re-expression [PMID:8636214]; the armadillo repeats act as a single structural unit required for axonemal assembly [PMID:10913963]. The human and mouse proteins localize to the sperm flagellum and decorate cytoplasmic microtubules in transfected cells [PMID:10493827, PMID:10684790]. SPAG6 builds a central-apparatus interaction network, binding PF20/SPAG16 and PF6 and being required for the stable assembly of both partners in the axoneme [PMID:12391165, PMID:15827353]. Beyond the axoneme, SPAG6 associates with the centrosome and cytoplasmic microtubules to control ciliogenesis, basal-foot orientation and epithelial planar cell polarity, with loss causing reduced and disorganized ciliary beating and loss of Vangl2 polarity [PMID:25333478], and to support centrosome polarization during immunological synapse formation [PMID:27169488]. SPAG6 regulates cell proliferation, adhesion, motility and microtubule acetylation, and its deletion produces supernumerary centrosomes and defective primary cilia [PMID:26585507]. During spermiogenesis it stabilizes acrosomal SPINK2 and modulates AKAP4 and SPAG16L levels, and compound loss with its paralogue causes infertility with defective acrosome, manchette and flagellar sheath formation [PMID:32216237, PMID:40777416]. In hematological malignancies SPAG6 acts as a pro-survival, pro-proliferative factor, suppressing apoptosis through PTEN/PI3K/AKT, TRAIL/FADD, AMPK/mTOR/ULK1 autophagy, AKT/FOXO cell-cycle, and ROS/GSTP1/JNK signaling, and its expression is controlled by promoter methylation and a STAT1 feedback loop [PMID:25405588, PMID:28393201, PMID:29749435, PMID:33000212, PMID:37681349, PMID:39508041].","teleology":[{"year":1996,"claim":"Established the founding mechanistic role: SPAG6's orthologue PF16 is a dedicated structural component of the C1 microtubule whose absence collapses the central apparatus and abolishes motility.","evidence":"Insertional mutagenesis, transformation rescue, and immunogold EM in Chlamydomonas","pmids":["8636214"],"confidence":"High","gaps":["Did not resolve which armadillo repeats mediate assembly","No direct binding partners identified at this stage"]},{"year":2000,"claim":"Defined the structural logic of assembly by showing the armadillo repeats function as a single unit, with the first repeat necessary but not sufficient and the C-terminus dispensable.","evidence":"Site-directed mutagenesis and deletion-construct rescue in pf16 mutants","pmids":["10913963"],"confidence":"High","gaps":["No atomic structure of the repeat unit","Binding determinants for partner proteins not mapped"]},{"year":1999,"claim":"Extended the central-apparatus role to mammals by showing human and mouse SPAG6 localize to the sperm flagellum and associate with microtubules.","evidence":"cDNA cloning and immunofluorescence on permeabilized sperm and transfected COS-1 cells","pmids":["10493827","10684790"],"confidence":"Medium","gaps":["Localization in somatic motile cilia not addressed","No functional knockout yet"]},{"year":2005,"claim":"Resolved how SPAG6 organizes the central apparatus by demonstrating it bridges PF20/SPAG16 and PF6 and is required for their stable assembly, defining a PF6–SPAG6–PF20 network.","evidence":"Yeast two-hybrid, co-localization in CHO cells, and immunoblot of Spag6-knockout mouse sperm","pmids":["12391165","15827353"],"confidence":"High","gaps":["Stoichiometry and architecture of the network not defined","Whether interactions are direct in the assembled axoneme unconfirmed"]},{"year":2014,"claim":"Broadened SPAG6's role beyond beating to ciliogenesis and tissue polarity, and to centrosome-dependent immune cell function.","evidence":"Spag6-knockout mouse analysis of ependymal/tracheal cilia, Vangl2 distribution, and lymphocyte immunological synapse assays","pmids":["25333478","27169488"],"confidence":"High","gaps":["Molecular link between SPAG6 and Vangl2 PCP machinery unresolved","Mechanism of centrosome polarization control not defined"]},{"year":2015,"claim":"Identified SPAG6 as a cytoplasmic regulator of centrosome number, cell morphology, motility, and microtubule acetylation, and as a stabilizer of partner proteins in distinct cell types.","evidence":"Spag6-knockout MEF assays with rescue, plus co-IP of prestin in outer hair cells and neuronal migration assays","pmids":["26585507","25748314","26130477"],"confidence":"Medium","gaps":["Direct biochemical basis for tubulin acetylation change unknown","Mechanism of centrosome amplification not established"]},{"year":2019,"claim":"Showed SPAG6 stabilizes acrosomal SPINK2 during spermiogenesis, extending its scaffolding role to acrosome formation.","evidence":"Yeast two-hybrid, co-localization, and knockout-mouse immunoblot/immunofluorescence","pmids":["32216237"],"confidence":"Medium","gaps":["Functional consequence of SPINK2 loss for fertility addressed only later","Direct binding interface not mapped"]},{"year":2020,"claim":"Reframed SPAG6 as a pro-survival oncogenic factor in hematological malignancies, converging on PTEN/PI3K/AKT, TRAIL/FADD, autophagy, and cell-cycle control of apoptosis.","evidence":"shRNA/siRNA knockdown and overexpression with pharmacological pathway rescue in MDS, leukemia and lymphoma lines plus xenografts","pmids":["25405588","28393201","29749435","30835546","32537026","33000212"],"confidence":"Medium","gaps":["How an axonemal scaffold engages these signaling pathways mechanistically is unclear","Most readouts are pathway correlations from a single lab lineage"]},{"year":2022,"claim":"Connected SPAG6 expression to epigenetic and transcriptional control and to a direct cytoskeletal motor partner driving oncogenic signaling.","evidence":"Co-IP/mass spectrometry of MYO1D, fractionation, and DNMT3b promoter-methylation/luciferase assays","pmids":["35667090","36398260"],"confidence":"Medium","gaps":["Whether MYO1D translocation is direct or scaffold-dependent unresolved","Context-dependent tumor-suppressor vs oncogene roles not reconciled"]},{"year":2023,"claim":"Established a STAT1–SPAG6 positive feedback loop and additional apoptosis axes, deepening the transcriptional regulation of SPAG6 in malignancy.","evidence":"ChIP and dual-luciferase assays plus knockdown/overexpression in MPN, and TGF-β/Smad pathway analysis in B-ALL","pmids":["37681349","38147275"],"confidence":"Medium","gaps":["B-ALL TGF-β/Smad link is low-confidence pathway correlation","Generality of STAT1 feedback across cell types untested"]},{"year":2025,"claim":"Distinguished SPAG6 from its paralogue SPAG6L, showing antagonistic effects on tubulin acetylation and differential SPINK2 affinity, and confirmed central-apparatus scaffolding by cryoEM.","evidence":"Compound knockout mouse genetics with binding-affinity assays (preprint) and cryoEM of the T. brucei central apparatus (preprint)","pmids":["40777416"],"confidence":"Medium","gaps":["Both findings are preprints awaiting peer review","cryoEM is in a non-mammalian organism without mutagenesis validation","DUSP1/MAPK interaction in myeloma remains low-confidence"]},{"year":null,"claim":"How a structural axonemal scaffold mechanistically rewires intracellular signaling pathways (PI3K/AKT, JNK, MAPK) in non-ciliated cancer cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct biochemical link between SPAG6 scaffolding and PTEN/AKT regulation","Reconciliation of tumor-suppressor vs oncogene context not achieved","No structure of mammalian SPAG6 or its partner complexes"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,3,4]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,27]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,5,15]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,21]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[7,9]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[6,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,13,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[17,18,23]}],"complexes":["axoneme central apparatus (C1 microtubule)"],"partners":["SPAG16","PF6","SPINK2","MYO1D","PRESTIN","TAC1","DUSP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75602","full_name":"Sperm-associated antigen 6","aliases":["Protein PF16 homolog","Repro-SA-1","Sperm flagellar protein"],"length_aa":509,"mass_kda":55.5,"function":"Important for structural integrity of the central apparatus in the sperm tail and for flagellar motility","subcellular_location":"Cytoplasm, cytoskeleton; Cell projection, cilium, flagellum; Cytoplasm, cytoskeleton, cilium axoneme","url":"https://www.uniprot.org/uniprotkb/O75602/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPAG6","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPAG6","total_profiled":1310},"omim":[{"mim_id":"619712","title":"SPERMATOGENIC FAILURE 65; SPGF65","url":"https://www.omim.org/entry/619712"},{"mim_id":"619144","title":"SPERMATOGENIC FAILURE 49; SPGF49","url":"https://www.omim.org/entry/619144"},{"mim_id":"618735","title":"TETRATRICOPEPTIDE REPEAT DOMAIN-CONTAINING PROTEIN 29; TTC29","url":"https://www.omim.org/entry/618735"},{"mim_id":"618661","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 70; CFAP70","url":"https://www.omim.org/entry/618661"},{"mim_id":"618433","title":"SPERMATOGENIC FAILURE 38; SPGF38","url":"https://www.omim.org/entry/618433"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Calyx","reliability":"Supported"},{"location":"Mid piece","reliability":"Supported"},{"location":"Principal piece","reliability":"Supported"},{"location":"End piece","reliability":"Supported"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":94.1},{"tissue":"fallopian 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Chlamydomonas) contains eight contiguous armadillo repeats and localizes specifically to the C1 microtubule of the central apparatus in flagella; loss of PF16 results in absence of the C1 microtubule and flagellar paralysis, and rescue by transformation restores wild-type motility and ultrastructure.\",\n      \"method\": \"Insertional mutagenesis, cDNA cloning, transformation rescue, immunofluorescence and immunogold electron microscopy with anti-PF16 antibody\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genetic rescue with defined phenotypic readout, direct localization by immunogold EM, replicated by multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"8636214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Functional domain analysis of PF16 demonstrated that the first armadillo repeat is necessary but not sufficient for assembly into the axoneme; the C-terminal 122 amino acids are dispensable for assembly and motility; and the armadillo repeats function as a single structural unit required for PF16 assembly.\",\n      \"method\": \"Site-directed mutagenesis of PF16 locus alleles, deletion constructs transformed into pf16 mutants, motility rescue and ultrastructural analysis\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vivo mutagenesis with functional rescue readout, multiple deletion constructs tested systematically in a single study\",\n      \"pmids\": [\"10913963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human SPAG6 protein, containing eight contiguous armadillo repeats homologous to Chlamydomonas PF16, localizes to the tails of permeabilized human sperm, consistent with a role in the axoneme central apparatus.\",\n      \"method\": \"cDNA cloning, immunofluorescence with anti-SPAG6 antibody on permeabilized human sperm\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment; single lab but corroborated by prior Chlamydomonas work and subsequent mammalian studies\",\n      \"pmids\": [\"10493827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Mouse Spag6 protein (murine SPAG6) localizes to the principal piece of permeabilized sperm tails and co-localizes with microtubules when expressed in COS-1 cells, establishing it as a microtubule-associated axonemal central apparatus protein.\",\n      \"method\": \"Antipeptide antibody immunofluorescence on mouse sperm and COS-1 cells transfected with Spag6 expression construct\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by immunofluorescence in two cell systems, single lab\",\n      \"pmids\": [\"10684790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Mammalian PF20 (SPAG16) interacts with SPAG6 via its WD repeat domain, as shown by yeast two-hybrid assay; when co-expressed in CHO cells, PF20 co-localizes with SPAG6 on polymerized microtubules; and PF20 protein is markedly reduced in sperm of Spag6-deficient mice, indicating SPAG6 is required for PF20 stability or assembly in the axoneme.\",\n      \"method\": \"Yeast two-hybrid, co-expression in CHO cells with fluorescence microscopy, immunoblotting of Spag6-knockout mouse sperm\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal evidence from yeast two-hybrid, co-localization, and in vivo knockout, multiple orthogonal methods across two proteins\",\n      \"pmids\": [\"12391165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mammalian PF6 (the orthologue of Chlamydomonas PF6, localized to the C1 1a projection) interacts with SPAG6 via a defined fragment, demonstrated by yeast two-hybrid and co-localization in transfected cells; a PF6 fragment corresponding to the SPAG6-binding domain is absent from sperm of SPAG6-deficient mice, establishing PF6–SPAG6–PF20 as a network linking C1 microtubule projections to the central bridge in the axoneme.\",\n      \"method\": \"Yeast two-hybrid, co-transfection and co-localization in mammalian cells, immunoblotting of Spag6-knockout mouse sperm\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (two-hybrid, co-localization, in vivo knockout), single lab but rigorous\",\n      \"pmids\": [\"15827353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In Spag6-deficient mice, ependymal and tracheal cilia show reduced beat frequency, unsynchronized beating, reduced cilia density, and randomized basal foot orientation; planar cell polarity protein Vangl2 distribution is lost in tracheal epithelial cells, demonstrating that SPAG6 regulates not only ciliary motility but also ciliogenesis, axoneme orientation, and epithelial planar cell polarity.\",\n      \"method\": \"Spag6 knockout mouse analysis: high-speed video microscopy of cilia beat, scanning/transmission electron microscopy, immunofluorescence for basal feet orientation and Vangl2 distribution\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with multiple defined cellular phenotypes, orthogonal methods (video microscopy, EM, IF), single lab\",\n      \"pmids\": [\"25333478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPAG6 deficiency in mice leads to impaired immunological synapse formation due to loss of centrosome polarization and failure of actin clearance at the synaptic cleft in lymphocytes, resulting in defective CTL function and reduced humoral immunity (germinal centers, class-switched antibody); SPAG6 is associated with the centrosome in lymphocytes.\",\n      \"method\": \"Bone marrow reconstitution, immunofluorescence for centrosome polarization and actin distribution in lymphocytes, CTL killing assays, antibody production assays in Spag6-knockout mice\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with specific mechanistic phenotype (centrosome polarization), multiple immune functional readouts, single lab\",\n      \"pmids\": [\"27169488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPAG6 silencing in myeloid leukemia cells (SKM-1 and K562) induces apoptosis via activation of caspase-3, -9, and -8, and upregulates p53 and PTEN expression at mRNA and protein levels; SPAG6-shRNA xenograft mice showed inhibited tumor growth and increased apoptosis.\",\n      \"method\": \"shRNA lentiviral knockdown, flow cytometry, CCK-8 assay, Western blot, RT-PCR, NOD/SCID xenograft model\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with multiple readouts (apoptosis, caspase activation, p53/PTEN upregulation) in vitro and in vivo, single lab\",\n      \"pmids\": [\"25405588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Spag6-deficient mouse embryonic fibroblasts proliferate more slowly, have larger surface area, reduced motility (chemotaxis and wound healing), reduced adhesion with non-polarized F-actin distribution, multiple centrosomes, reduced primary cilia formation, and increased sensitivity to paclitaxel; re-expression of SPAG6 rescues abnormal cell morphology; SPAG6 selectively increases acetylated tubulin levels.\",\n      \"method\": \"MEF isolation from Spag6-knockout embryos, proliferation assays, wound healing/chemotaxis assays, immunofluorescence for F-actin and tubulin, primary cilia quantification, rescue by SPAG6 re-expression\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with rescue, multiple orthogonal cellular assays, defined molecular correlate (acetylated tubulin), single lab\",\n      \"pmids\": [\"26585507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Overexpression of Spag6 in cortical neurons delays neuronal migration rate and decreases neurite number and length, as assayed by in utero electroporation, identifying a role for SPAG6 in regulating neuronal migration through cytoskeletal remodeling.\",\n      \"method\": \"In utero electroporation of mouse embryonic cortex, immunofluorescence and quantitative analysis of neuronal position and neurite morphology\",\n      \"journal\": \"Journal of molecular neuroscience : MN\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain-of-function with specific phenotypic readout in vivo, single lab, single method\",\n      \"pmids\": [\"26130477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPAG6 co-immunoprecipitates with prestin in outer hair cells (OHCs); Spag6-knockout mice show reduced prestin expression at both protein and mRNA levels and abnormal OHC morphology, indicating SPAG6 is required for prestin stability and normal OHC mechanosensory function.\",\n      \"method\": \"Co-immunoprecipitation, Western blot, real-time PCR, immunofluorescence in Spag6-knockout mice\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP demonstrates interaction, KO shows functional consequence, single lab\",\n      \"pmids\": [\"25748314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPAG6 regulates apoptosis in SKM-1 MDS cells via the TRAIL signaling pathway: SPAG6 knockdown activates the TRAIL pathway and increases interaction between FADD and TRAIL death receptors, while high SPAG6 expression suppresses TRAIL signaling; SPAG6 does not affect expression of TRAIL death receptors except FADD.\",\n      \"method\": \"shRNA lentiviral knockdown, flow cytometry, CCK-8 assay, Western blot, co-immunoprecipitation for FADD–death receptor interaction\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with pathway-specific mechanistic readouts and co-IP, single lab\",\n      \"pmids\": [\"28393201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPAG6 silencing in SKM-1 cells increases PTEN expression and reduces AKT phosphorylation, leading to apoptosis via Mcl-1 downregulation, cytochrome c release, and increased caspase-9; the PI3K inhibitor LY294002 synergistically enhances this apoptosis; SPAG6-regulated PTEN expression is caspase-independent; SPAG6 knockdown is associated with DNMT1 downregulation, suggesting SPAG6 may control PTEN expression via DNA methylation.\",\n      \"method\": \"shRNA lentiviral knockdown, Western blot, flow cytometry, PI3K inhibitor treatment, pan-caspase inhibitor treatment, in vivo xenograft model\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with pharmacological validation of pathway, multiple readouts, single lab\",\n      \"pmids\": [\"29749435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPAG6 knockdown in SKM-1 cells inhibits cell proliferation by mediating G1-to-S cell cycle arrest, upregulating p27Kip1, and regulating the AKT/FOXO signaling pathway.\",\n      \"method\": \"shRNA knockdown, CCK-8, FACS cell cycle analysis, RT-qPCR, Western blot\",\n      \"journal\": \"DNA and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with defined cell cycle and pathway readout, single lab\",\n      \"pmids\": [\"30835546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPAG6 interacts with SPINK2 (serine protease inhibitor Kazal-type 2) in the acrosome of round spermatids; SPAG6 is expressed in the acrosome during spermatogenesis (postnatal days 16–28); in SPAG6-knockout mice, SPINK2 expression and acrosomal localization are absent, indicating SPAG6 stabilizes SPINK2 during acrosome formation.\",\n      \"method\": \"Yeast two-hybrid, co-localization in CHO cells by immunofluorescence, Western blot and immunofluorescence in Spag6-knockout mice\",\n      \"journal\": \"Zhonghua nan ke xue = National journal of andrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid plus co-localization plus in vivo KO, single lab\",\n      \"pmids\": [\"32216237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG6 silencing in SKM-1 cells induces autophagic cell death via the AMPK/mTOR/ULK1 pathway: knockdown upregulates p-AMPK and p-ULK1 and downregulates p-mTOR; autophagy inhibition with chloroquine or 3-methyladenine decreases SPAG6-knockdown-mediated apoptosis; AMPK inhibition (Compound C) attenuates both autophagy and apoptosis.\",\n      \"method\": \"shRNA lentiviral knockdown, Western blot, flow cytometry, autophagy inhibitor and AMPK inhibitor pharmacological experiments\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with pharmacological pathway validation, single lab\",\n      \"pmids\": [\"32537026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG6 promotes proliferation and inhibits apoptosis in Burkitt lymphoma cells via the PTEN/PI3K/AKT pathway: SPAG6 knockdown increases PTEN and reduces p-AKT, while PTEN siRNA or PTEN inhibitor SF1670 reverses the anti-proliferative and pro-apoptotic effects of SPAG6 depletion both in vitro and in vivo.\",\n      \"method\": \"shRNA/siRNA knockdown and overexpression, Western blot, flow cytometry, PTEN inhibitor treatment, in vivo xenograft\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain- and loss-of-function with pharmacological rescue of PTEN, in vitro and in vivo, single lab\",\n      \"pmids\": [\"33000212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPAG6 interacts with MYO1D (myosin 1D) as identified by co-immunoprecipitation and mass spectrometry; SPAG6 overexpression promotes translocation of MYO1D from cytosol to cell membrane, upregulating EGFR family expression and thereby activating PI3K/AKT and ERK signaling to promote AML cell proliferation and migration.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, Western blot for MYO1D localization (membrane vs. cytosol fractionation), in vitro proliferation/migration assays, in vivo xenograft\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS for interaction, subcellular fractionation for localization change, in vitro and in vivo functional validation, single lab\",\n      \"pmids\": [\"35667090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPAG6 knockdown in MPN cell lines promotes apoptosis, reduces G1-to-S cell cycle progression, and is associated with downregulated STAT1; forced SPAG6 expression promotes clone formation and G1-to-S progression; chromatin immunoprecipitation shows STAT1 binds the SPAG6 promoter, and dual-luciferase assay shows STAT1 promotes SPAG6 transcription, establishing a positive feedback between SPAG6 and STAT1.\",\n      \"method\": \"shRNA knockdown, overexpression, FACS cell cycle analysis, ChIP assay, dual-luciferase reporter assay, Western blot\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase assays establish STAT1–SPAG6 transcriptional relationship; functional KD/OE with defined pathway, single lab\",\n      \"pmids\": [\"37681349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPAG6 knockdown in B-ALL cell lines inhibits proliferation and promotes apoptosis via the TGF-β/Smad signaling pathway.\",\n      \"method\": \"Lentiviral knockdown, cell proliferation assays, flow cytometry for apoptosis, Western blot for TGF-β/Smad pathway components\",\n      \"journal\": \"International journal of hematology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — loss-of-function with pathway correlation but limited mechanistic validation, single lab, single method type\",\n      \"pmids\": [\"38147275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TAC1 protein interacts with SPAG6 as shown by yeast two-hybrid; when co-transfected with SPAG6 in CHO cells, TAC1 localizes to microtubules rather than remaining diffusely cytoplasmic, indicating SPAG6 directs TAC1 to microtubules.\",\n      \"method\": \"Yeast two-hybrid, co-transfection and immunofluorescence co-localization in CHO and COS-1 cells, Western blot\",\n      \"journal\": \"Zhonghua nan ke xue = National journal of andrology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, yeast two-hybrid plus co-localization only, limited functional follow-up\",\n      \"pmids\": [\"29282908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DNMT3b-mediated hypermethylation of the SPAG6 promoter leads to SPAG6 downregulation in lung squamous cell carcinoma; restored SPAG6 expression inhibits STAT1 and STAT3 transcription, reversing JAK/STAT pathway activation and suppressing malignant cell phenotype.\",\n      \"method\": \"Bisulfite sequencing, DNMT3b functional assays, dual-luciferase assay for STAT1/STAT3 transcription, CCK-8/EdU/Transwell assays\",\n      \"journal\": \"American journal of translational research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — methylation writer identified (DNMT3b), luciferase assay for downstream transcriptional effect, single lab\",\n      \"pmids\": [\"36398260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPAG6 overexpression in AML cells reduces ROS and malondialdehyde levels and promotes GSTP1 expression; elevated GSTP1 forms a complex with JNK, reducing p-JNK and inhibiting JNK pathway activation, thereby attenuating the pro-apoptotic effects of daunorubicin; SPAG6 knockdown diminishes mitochondrial membrane potential and increases apoptosis.\",\n      \"method\": \"TMT proteomics after SPAG6 knockdown, CCK-8, flow cytometry for ROS and apoptosis, Western blot, in vivo xenograft, IHC\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — proteomics plus functional validation of ROS/GSTP1/JNK axis, in vitro and in vivo, single lab\",\n      \"pmids\": [\"39508041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPAG6 directly interacts with DUSP1 (dual-specificity phosphatase 1) in multiple myeloma cells, and SPAG6 modulates downstream MAPK/ERK pathway activity by regulating DUSP1 activity; SPAG6 overexpression promotes proliferation, migration, and apoptosis resistance, while knockdown has opposite effects.\",\n      \"method\": \"Transcriptome sequencing, co-immunoprecipitation (implied from 'directly interacts'), RNA interference, overexpression, cell proliferation/apoptosis/migration assays\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — interaction and pathway modulation reported but mechanistic detail of DUSP1 regulation is limited in abstract, single lab\",\n      \"pmids\": [\"40535772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Both SPAG6 and SPAG6L bind SPINK2, but SPAG6 has approximately 10-fold higher binding affinity than SPAG6L; SPAG6 modulates testicular AKAP4 and SPAG16L levels; compound Spag6−/−;Spag6l+/− males are infertile with defective spermiogenesis (abnormal chromatin condensation, malformed acrosome and manchette, disorganized mitochondrial sheath and fibrous sheath); SPAG6 suppresses tubulin acetylation whereas SPAG6L enhances it, indicating antagonistic roles in microtubule regulation.\",\n      \"method\": \"Compound knockout mouse genetics, histological and ultrastructural analysis, binding affinity assays for SPINK2, Western blot for AKAP4 and SPAG16L, acetylated tubulin immunoblotting\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — compound KO genetics with multiple ultrastructural and molecular phenotypes, binding affinity comparison, preprint not yet peer-reviewed\",\n      \"pmids\": [\"40777416\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The SPAG6 c.900 T>C variant in exon 7 generates a new splice acceptor site that inhibits exon 7 skipping, thereby promoting Sertoli cell growth and maintaining normal blood-testis barrier function; the variant is associated with semen quality traits in pigs.\",\n      \"method\": \"RNA-seq analysis, Sanger sequencing, splice site reporter assay, Sertoli cell culture assays for growth and blood-testis barrier function\",\n      \"journal\": \"Animal genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — splice site creation mechanistically validated in cell culture with functional blood-testis barrier readout, single lab\",\n      \"pmids\": [\"37211688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CryoEM of the Trypanosoma brucei flagellum central apparatus identified PF16 (SPAG6 orthologue) as a structural scaffold critical for central apparatus assembly and axonemal asymmetry; PF16 is stably associated with the C1/C2 microtubules.\",\n      \"method\": \"Cryogenic electron microscopy (cryoEM) structural analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — high-quality structural method (cryoEM), but in a non-mammalian organism (T. brucei) and preprint, not directly validated by mutagenesis\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SPAG6 is an armadillo-repeat-containing protein that functions as a structural scaffold on the C1 microtubule of the axoneme central apparatus, where it stabilizes the C1 microtubule and interacts with PF20 (SPAG16), PF6, SPINK2, MYO1D, prestin, and TAC1 to maintain axoneme integrity and regulate flagellar/ciliary motility; beyond its canonical axonemal role, SPAG6 associates with cytoplasmic microtubules and the centrosome to regulate ciliogenesis, planar cell polarity, epithelial cell polarity, cell proliferation, migration, and immunological synapse formation, and in hematological malignancies it modulates apoptosis via TRAIL, PTEN/PI3K/AKT, AMPK/mTOR/ULK1, AKT/FOXO, and ROS/JNK/GSTP1 signaling pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPAG6 is an armadillo-repeat protein that functions as a structural scaffold of the central apparatus of motile cilia and flagella, where it stabilizes the C1 microtubule and organizes the projections that confer axonemal asymmetry [#0, #27]. First defined through the Chlamydomonas orthologue PF16, SPAG6 localizes specifically to the C1 microtubule, and its loss eliminates the C1 microtubule and paralyzes the flagellum, a phenotype rescued by re-expression [#0]; the armadillo repeats act as a single structural unit required for axonemal assembly [#1]. The human and mouse proteins localize to the sperm flagellum and decorate cytoplasmic microtubules in transfected cells [#2, #3]. SPAG6 builds a central-apparatus interaction network, binding PF20/SPAG16 and PF6 and being required for the stable assembly of both partners in the axoneme [#4, #5]. Beyond the axoneme, SPAG6 associates with the centrosome and cytoplasmic microtubules to control ciliogenesis, basal-foot orientation and epithelial planar cell polarity, with loss causing reduced and disorganized ciliary beating and loss of Vangl2 polarity [#6], and to support centrosome polarization during immunological synapse formation [#7]. SPAG6 regulates cell proliferation, adhesion, motility and microtubule acetylation, and its deletion produces supernumerary centrosomes and defective primary cilia [#9]. During spermiogenesis it stabilizes acrosomal SPINK2 and modulates AKAP4 and SPAG16L levels, and compound loss with its paralogue causes infertility with defective acrosome, manchette and flagellar sheath formation [#15, #25]. In hematological malignancies SPAG6 acts as a pro-survival, pro-proliferative factor, suppressing apoptosis through PTEN/PI3K/AKT, TRAIL/FADD, AMPK/mTOR/ULK1 autophagy, AKT/FOXO cell-cycle, and ROS/GSTP1/JNK signaling, and its expression is controlled by promoter methylation and a STAT1 feedback loop [#8, #12, #13, #17, #19, #23].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established the founding mechanistic role: SPAG6's orthologue PF16 is a dedicated structural component of the C1 microtubule whose absence collapses the central apparatus and abolishes motility.\",\n      \"evidence\": \"Insertional mutagenesis, transformation rescue, and immunogold EM in Chlamydomonas\",\n      \"pmids\": [\"8636214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which armadillo repeats mediate assembly\", \"No direct binding partners identified at this stage\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined the structural logic of assembly by showing the armadillo repeats function as a single unit, with the first repeat necessary but not sufficient and the C-terminus dispensable.\",\n      \"evidence\": \"Site-directed mutagenesis and deletion-construct rescue in pf16 mutants\",\n      \"pmids\": [\"10913963\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic structure of the repeat unit\", \"Binding determinants for partner proteins not mapped\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Extended the central-apparatus role to mammals by showing human and mouse SPAG6 localize to the sperm flagellum and associate with microtubules.\",\n      \"evidence\": \"cDNA cloning and immunofluorescence on permeabilized sperm and transfected COS-1 cells\",\n      \"pmids\": [\"10493827\", \"10684790\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Localization in somatic motile cilia not addressed\", \"No functional knockout yet\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved how SPAG6 organizes the central apparatus by demonstrating it bridges PF20/SPAG16 and PF6 and is required for their stable assembly, defining a PF6\\u2013SPAG6\\u2013PF20 network.\",\n      \"evidence\": \"Yeast two-hybrid, co-localization in CHO cells, and immunoblot of Spag6-knockout mouse sperm\",\n      \"pmids\": [\"12391165\", \"15827353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the network not defined\", \"Whether interactions are direct in the assembled axoneme unconfirmed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Broadened SPAG6's role beyond beating to ciliogenesis and tissue polarity, and to centrosome-dependent immune cell function.\",\n      \"evidence\": \"Spag6-knockout mouse analysis of ependymal/tracheal cilia, Vangl2 distribution, and lymphocyte immunological synapse assays\",\n      \"pmids\": [\"25333478\", \"27169488\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between SPAG6 and Vangl2 PCP machinery unresolved\", \"Mechanism of centrosome polarization control not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified SPAG6 as a cytoplasmic regulator of centrosome number, cell morphology, motility, and microtubule acetylation, and as a stabilizer of partner proteins in distinct cell types.\",\n      \"evidence\": \"Spag6-knockout MEF assays with rescue, plus co-IP of prestin in outer hair cells and neuronal migration assays\",\n      \"pmids\": [\"26585507\", \"25748314\", \"26130477\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical basis for tubulin acetylation change unknown\", \"Mechanism of centrosome amplification not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed SPAG6 stabilizes acrosomal SPINK2 during spermiogenesis, extending its scaffolding role to acrosome formation.\",\n      \"evidence\": \"Yeast two-hybrid, co-localization, and knockout-mouse immunoblot/immunofluorescence\",\n      \"pmids\": [\"32216237\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of SPINK2 loss for fertility addressed only later\", \"Direct binding interface not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Reframed SPAG6 as a pro-survival oncogenic factor in hematological malignancies, converging on PTEN/PI3K/AKT, TRAIL/FADD, autophagy, and cell-cycle control of apoptosis.\",\n      \"evidence\": \"shRNA/siRNA knockdown and overexpression with pharmacological pathway rescue in MDS, leukemia and lymphoma lines plus xenografts\",\n      \"pmids\": [\"25405588\", \"28393201\", \"29749435\", \"30835546\", \"32537026\", \"33000212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How an axonemal scaffold engages these signaling pathways mechanistically is unclear\", \"Most readouts are pathway correlations from a single lab lineage\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected SPAG6 expression to epigenetic and transcriptional control and to a direct cytoskeletal motor partner driving oncogenic signaling.\",\n      \"evidence\": \"Co-IP/mass spectrometry of MYO1D, fractionation, and DNMT3b promoter-methylation/luciferase assays\",\n      \"pmids\": [\"35667090\", \"36398260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MYO1D translocation is direct or scaffold-dependent unresolved\", \"Context-dependent tumor-suppressor vs oncogene roles not reconciled\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a STAT1\\u2013SPAG6 positive feedback loop and additional apoptosis axes, deepening the transcriptional regulation of SPAG6 in malignancy.\",\n      \"evidence\": \"ChIP and dual-luciferase assays plus knockdown/overexpression in MPN, and TGF-\\u03b2/Smad pathway analysis in B-ALL\",\n      \"pmids\": [\"37681349\", \"38147275\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"B-ALL TGF-\\u03b2/Smad link is low-confidence pathway correlation\", \"Generality of STAT1 feedback across cell types untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Distinguished SPAG6 from its paralogue SPAG6L, showing antagonistic effects on tubulin acetylation and differential SPINK2 affinity, and confirmed central-apparatus scaffolding by cryoEM.\",\n      \"evidence\": \"Compound knockout mouse genetics with binding-affinity assays (preprint) and cryoEM of the T. brucei central apparatus (preprint)\",\n      \"pmids\": [\"40777416\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Both findings are preprints awaiting peer review\", \"cryoEM is in a non-mammalian organism without mutagenesis validation\", \"DUSP1/MAPK interaction in myeloma remains low-confidence\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a structural axonemal scaffold mechanistically rewires intracellular signaling pathways (PI3K/AKT, JNK, MAPK) in non-ciliated cancer cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biochemical link between SPAG6 scaffolding and PTEN/AKT regulation\", \"Reconciliation of tumor-suppressor vs oncogene context not achieved\", \"No structure of mammalian SPAG6 or its partner complexes\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 27]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 5, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 21]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [7, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 13, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [17, 18, 23]}\n    ],\n    \"complexes\": [\"axoneme central apparatus (C1 microtubule)\"],\n    \"partners\": [\"SPAG16\", \"PF6\", \"SPINK2\", \"MYO1D\", \"prestin\", \"TAC1\", \"DUSP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}