{"gene":"FOXO3","run_date":"2026-06-09T23:54:44","timeline":{"discoveries":[{"year":2001,"finding":"SGK1 phosphorylates FOXO3a (FKHRL1) at Ser-315 (with preference over Akt), while Akt preferentially phosphorylates Ser-253; both kinases phosphorylate Thr-32. Phosphorylation of all three sites by SGK1 and Akt in concert suppresses FOXO3a-dependent transcription, leading to nuclear exclusion and inhibition of cell cycle arrest and apoptosis. SGK1 acts downstream of PI3K, like Akt, and promotes cell survival in part through this mechanism.","method":"In vitro kinase assays, site-directed mutagenesis of phosphorylation sites, overexpression and dominant-negative constructs, transcriptional reporter assays, cell survival assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis at three defined sites, multiple orthogonal methods, replicated across conditions","pmids":["11154281"],"is_preprint":false},{"year":2001,"finding":"TGF-β induces phosphorylation and cytoplasmic retention of FOXO3a (FKHRL1) in mammary epithelial cells via PI3K/Akt. A triple Akt-site mutant of FKHRL1 (TM-FKHRL1) that cannot be phosphorylated by Akt fails to translocate to the cytoplasm in response to TGF-β, and dominant-negative Akt blocks TGF-β-induced FKHRL1 nuclear exclusion, phosphorylation, and transcriptional suppression. FKHRL1-driven apoptosis requires its transactivation domain.","method":"Triple phosphosite mutant transfection, dominant-negative Akt, PI3K inhibitor (LY294002), nuclear exclusion assays, transcriptional reporter assays, apoptosis assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of all three Akt sites, dominant-negative Akt, PI3K inhibitor, multiple orthogonal methods in single lab","pmids":["11694570"],"is_preprint":false},{"year":2001,"finding":"FOXO3a (FKHRL1) is expressed in human megakaryocytes and is phosphorylated by thrombopoietin (TPO) downstream of PI3K/Akt. Induced expression of constitutively active FKHRL1 causes G0/G1 cell cycle arrest in TPO-dependent megakaryocytic cells, identifying FKHRL1 as a downstream regulator of cell cycle in megakaryocytopoiesis.","method":"Tetracycline-inducible expression system, cell cycle analysis (flow cytometry), PI3K/Akt phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — inducible expression system with defined cell cycle readout, single lab, two methods","pmids":["11278373"],"is_preprint":false},{"year":2002,"finding":"NGF rapidly induces phosphorylation of FKHRL1 (FOXO3a) in PC12 cells via TrkA receptor and PI3K/Akt kinase (but not MAP kinase or S6p70 kinase). Constitutively active Akt and in vitro kinase assays confirmed Akt as the mediating kinase. This phosphorylation contributes to neurotrophin-mediated cell survival.","method":"Kinase inhibitors, constitutively active and dominant-negative Akt transfection, in vitro kinase assay, TrkA-expressing vs p75-expressing cell comparison, survival assays with PI3K inhibitor","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus multiple genetic tools, single lab","pmids":["11953455"],"is_preprint":false},{"year":2004,"finding":"In response to DNA damage, p53 activation leads to FOXO3a (FKHRL1) phosphorylation and cytoplasmic translocation via SGK1, which is induced by p53 in an ERK1/2-dependent post-translational manner. AKT is dispensable for this p53-dependent suppression of FKHRL1; siRNA knockdown of SGK1 significantly decreases FKHRL1 phosphorylation after DNA damage.","method":"siRNA knockdown of SGK1, pharmacological inhibitors, subcellular localization assays, Western blotting for phosphorylation, genetic analysis in p53-deficient cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown combined with pharmacological inhibitors and localization assays, single lab","pmids":["15383658"],"is_preprint":false},{"year":2006,"finding":"FOXO3a (FKHRL1) induces apoptosis in neuroblastoma cells through the mitochondrial pathway by transcriptionally activating the BH3-only proteins Noxa and Bim, which together are critical for cytochrome c release and caspase-dependent apoptosis. RNAi knockdown of either Noxa or Bim reduces FOXO3a-induced cell death. The death-receptor pathway (TRAIL/dnFADD) is not required, whereas Bcl2 overexpression protects, confirming the Bcl2-rheostat model.","method":"4-OH-tamoxifen-regulated FKHRL1(A3)ERtm transgene, RNAi knockdown of Noxa and Bim, dominant-negative FADD expression, Bcl2 overexpression, cytochrome c release assay, caspase activity","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional transgene, RNAi of specific targets, multiple orthogonal death assays, replicated with multiple genetic perturbations","pmids":["16888645"],"is_preprint":false},{"year":2006,"finding":"FOXO3a (FKHRL1) and FOXO1a bind insulin response sequences (IRSs) in the glucose-6-phosphatase (G6Pase) promoter cooperatively (two adjacent IRSs), and their binding correlates with insulin-mediated inhibition of basal G6Pase transcription. Insulin stimulates nuclear exclusion of FOXO1a/3a in a PI3K-dependent manner in granulosa cells.","method":"Gel retardation/binding assays, overexpression experiments, fusion gene transcription assays in multiple promoter contexts, PI3K inhibition","journal":"Molecular endocrinology (Baltimore, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct DNA binding assays with overexpression and promoter constructs, single lab, two orthogonal methods","pmids":["16840535"],"is_preprint":false},{"year":2007,"finding":"AGE-induced oxidative stress causes phosphorylation of FOXO3a (FKHRL1) in a redox-dependent and p66shc Ser-36-phosphorylation-dependent manner, leading to ~70% downregulation of MnSOD. AGER1 overexpression suppresses these pro-oxidant responses; AGER1 siRNA restores them. PI3K inhibitor (LY-294002) and N-acetylcysteine partially block FKHRL1 phosphorylation-driven MnSOD suppression.","method":"p66shc Ser-36 mutants, AGER1 overexpression/siRNA, PI3K inhibitor, antioxidant rescue, Western blotting","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined mutants and siRNA, single lab, multiple inhibitors","pmids":["18032526"],"is_preprint":false},{"year":2008,"finding":"FOXO3a interacts with the ATM FAT domain (C-terminal domain of FOXO3a binds FAT domain of ATM) and promotes ATM autophosphorylation at Ser-1981, leading to formation of ATM-pS1981 and γ-H2AX nuclear foci in response to DNA damage. Silencing FOXO3a abrogates ATM foci formation and DNA repair. FOXO3a promotes intra-S and G2/M checkpoints and facilitates DNA repair.","method":"Co-immunoprecipitation, domain-mapping, siRNA knockdown, immunofluorescence foci assays, cell cycle checkpoint analysis, DNA repair assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-mapped Co-IP, siRNA loss-of-function, multiple functional readouts (foci, checkpoints, repair), single rigorous study","pmids":["18344987"],"is_preprint":false},{"year":2008,"finding":"FoxO3a overexpression in muscle precursor cells (MPCs) impairs proliferation by preferentially inducing p27Kip1 promoter activity and protein expression (but not p21Waf/Cip1, which is decreased). FoxO3a overexpression does not affect retinoblastoma phosphorylation or cyclin D1, indicating it does not impair the early G1/S transition.","method":"Adenoviral FoxO3a overexpression, BrdU incorporation assay, promoter-luciferase assays for p27Kip1 and p21Waf/Cip1, Western blotting","journal":"Muscle & nerve","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression with promoter-luciferase and protein readouts, single lab","pmids":["17894357"],"is_preprint":false},{"year":2009,"finding":"FOXO3a (FKHRL1) represses BIRC5/Survivin transcription and protein expression. Conditional FKHRL1 activation sensitizes neuroblastoma cells to DNA-damaging agents (doxorubicin, etoposide). Transgenic Survivin rescues FKHRL1-induced apoptosis and prevents Bim/Bax mitochondrial accumulation, cytochrome c release, and loss of mitochondrial membrane potential. Survivin shRNA accelerates FKHRL1-induced apoptosis.","method":"4-OH-tamoxifen-regulated FKHRL1(A3)ERtm allele, retroviral Survivin shRNA, transgenic Survivin rescue, cytochrome c release, mitochondrial membrane potential assay, drug sensitivity assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional transgene, shRNA knockdown, transgenic rescue, multiple orthogonal mitochondrial readouts","pmids":["19211844"],"is_preprint":false},{"year":2009,"finding":"FoxO3 is required for neural stem cell (NSC) pool maintenance. FoxO3-/- adult mice have fewer NSCs in vivo, and NSCs from FoxO3-/- mice show decreased self-renewal and impaired multipotency. FoxO3 regulates a gene expression program in NSCs that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism.","method":"FoxO3 knockout mice, neurosphere assays (self-renewal), multipotency assays, gene expression profiling","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with in vivo and in vitro cellular phenotype, gene expression profiling, multiple orthogonal readouts","pmids":["19896443"],"is_preprint":false},{"year":2011,"finding":"During denervation-induced muscle atrophy, FoxO3 is progressively acetylated by the histone acetyltransferase p300 (demonstrated by co-immunoprecipitation). Acetylation causes cytosolic relocalization and proteasomal degradation of FoxO3, coinciding with downregulation of its target atrogin-1. Acetylation-mimicking FoxO3 mutants show decreased transcriptional activity and cytosolic localization; Lys-262 is critical for FoxO3 translocation. Acetylation thus negatively modulates FoxO3 activity.","method":"Co-immunoprecipitation (FoxO3-p300), acetylation-mimic and acetylation-resistant FoxO3 mutants, denervation mouse model, subcellular fractionation, proteasome inhibition","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP with defined acetyltransferase, site-directed mutagenesis of multiple lysines, in vivo denervation model, proteasome inhibition rescue","pmids":["22094330"],"is_preprint":false},{"year":2011,"finding":"TEAD1 directly binds the M-CAT element in the FoxO3a promoter and transcriptionally activates FoxO3a expression in skeletal muscle, as demonstrated by ChIP, EMSA, and luciferase reporter assays. Overexpression and inhibition experiments confirm TEAD1 as a positive regulator of FoxO3a.","method":"ChIP-on-chip, independent ChIP-PCR, EMSA, luciferase reporter assays, overexpression and inhibition experiments","journal":"BMC molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal DNA-binding methods (ChIP, EMSA, luciferase), single lab","pmids":["21211055"],"is_preprint":false},{"year":2012,"finding":"FOXO3 interacts with the ATM-Chk2-p53 complex (demonstrated by Co-IP), augments phosphorylation of all complex members, and induces nuclear foci formation upon DNA damage. FOXO3 is essential for DNA damage-induced apoptosis; conversely, FOXO3 requires ATM, Chk2, and phospho-p53 isoforms to trigger apoptosis. FOXO3 also contributes to chromatin retention of phosphorylated p53.","method":"Co-immunoprecipitation of FOXO3-ATM-Chk2-p53 complex, siRNA knockdown, nuclear foci immunofluorescence, apoptosis assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP of multi-protein complex, genetic epistasis by siRNA of multiple pathway members, multiple functional readouts","pmids":["22893124"],"is_preprint":false},{"year":2012,"finding":"Conditional FOXO3 activation induces caspase-8 expression in neuroblastoma without changing DNA methylation of the caspase-8 gene. Instead, FOXO3 induces phosphorylation of ATM, which then phosphorylates CREB; CREB phosphorylation is critical for FOXO3-mediated caspase-8 expression. The DNA-demethylating drug 5-azadC activates this FOXO3-ATM-CREB pathway to restore caspase-8 expression.","method":"Conditional FOXO3 transgene (ERtm), ATM inhibitor/knockdown, CREB inhibition, DNA methylation analysis (bisulfite), caspase-8 expression assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional transgene with mechanistic dissection using ATM and CREB inhibitors, single lab","pmids":["22493319"],"is_preprint":false},{"year":2016,"finding":"FOXO3a directly binds to the Keap1 promoter and drives basal Keap1 transcription. FoxO3 depletion reduces Keap1, thereby stabilizing and activating Nrf2. This FoxO3-Keap1-Nrf2 axis explains how AKT activation or TNF-α-mediated FoxO3 inactivation leads to Nrf2 induction. In vivo, FoxO3 deficiency potentiates tumor formation and cisplatin resistance in cholangiocarcinoma through Nrf2 activation.","method":"ChIP assay (FoxO3 on Keap1 promoter), siRNA knockdown, luciferase reporter, xenograft tumor model, ROS measurement","journal":"Hepatology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-confirmed promoter binding, siRNA KD with multiple epistasis experiments, in vivo xenograft model","pmids":["26857210"],"is_preprint":false},{"year":2016,"finding":"FOXO3a transcriptionally regulates BNIP3 expression in cardiomyocytes. Increased FOXO3a activity upregulates BNIP3, leading to increased mitochondrial Ca2+, decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis in adult cardiomyocytes. Dominant-negative FOXO3a (AAV9) in a rat HFpEF model decreases BNIP3, reverses adverse remodeling, and improves cardiac function.","method":"Dominant-negative FOXO3a adenovirus and AAV9 in vitro/in vivo, BNIP3 knockdown/overexpression, mitochondrial membrane potential assay, Ca2+ imaging, cardiac functional analysis (echocardiography)","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative construct in vitro and in vivo AAV delivery, multiple mitochondrial readouts, disease model rescue","pmids":["27694219"],"is_preprint":false},{"year":2019,"finding":"FoxO3 is regulated by prolyl hydroxylation: hypoxia inhibits prolyl hydroxylase (PHD)-mediated prolyl hydroxylation of FoxO3, preventing its degradation and leading to FoxO3 accumulation in renal tubular cells. Hypoxia-activated HIF-1α contributes to FoxO3 activation; tubular HIF-1α deletion reduces FoxO3 activation. Tubular FoxO3 deletion during AKI-to-CKD transition worsens structural/functional damage, decreases autophagy, and increases oxidative injury.","method":"Tubule-specific conditional FoxO3 knockout and HIF-1α knockout mice, prolyl hydroxylation assays, renal ischemia/reperfusion model, autophagy and oxidative stress markers","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO of FoxO3 and HIF-1α in same tissue, biochemical prolyl hydroxylation evidence, disease model with functional readouts","pmids":["30912765"],"is_preprint":false},{"year":2019,"finding":"FOXO3 directly transactivates SIRT6 by binding to its promoter region (confirmed by luciferase assay and ChIP). The FOXO3a-SIRT6 axis suppresses aerobic glycolysis (Warburg effect) in melanoma cells. SIRT6 knockdown or overexpression rescues the glycolytic effects of FOXO3a manipulation.","method":"ChIP assay, luciferase reporter assay, lentiviral SIRT6 knockdown/overexpression rescue, glycolysis assays (glucose uptake, lactate, Seahorse), mouse xenograft","journal":"International journal of oncology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP and luciferase confirm direct promoter binding, epistatic rescue experiments with multiple metabolic readouts","pmids":["32124950"],"is_preprint":false},{"year":2019,"finding":"FOXO3 directly binds the SIRT6 promoter in glioblastoma cells and transcriptionally activates SIRT6, thereby suppressing glycolysis (Warburg effect). FKHRL1 knockdown reduces SIRT6 expression and increases glycolysis; SIRT6 restoration reverses the glycolytic phenotype caused by FKHRL1 knockdown. This axis is confirmed both in vitro and in vivo.","method":"Luciferase assay, ChIP assay, siRNA knockdown, SIRT6 overexpression rescue, Seahorse glycolysis assay, xenograft model","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP and luciferase assays confirm direct binding; epistatic rescue experiments; in vivo validation","pmids":["31004738"],"is_preprint":false},{"year":2019,"finding":"Small molecules targeting the FOXO3 DNA-binding domain (DBD) physically interact with it (validated by NMR spectroscopy and docking). Compounds S9 and S9OX interfere with FOXO3 target promoter binding, gene transcription, and modulate the physiological transcriptional program in cancer cells, demonstrating the druggability of the FOXO3-DBD.","method":"Pharmacophore-modeling in silico screen, fluorescence polarization binding assay, NMR spectroscopy, docking studies, transcriptional target assays in human cells","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural validation of binding, in vitro fluorescence polarization assay, functional transcriptional readout, single lab","pmids":["31789593"],"is_preprint":false},{"year":2019,"finding":"FOXO3 directly transcribes PERK (eIF2AK3) as established by ChIP, siRNA knockdown, and overexpression assays, as well as validation in Foxo1/3/4-/- MEFs. Drug-resistant breast cancer cells with low FOXO3/PERK expression and high PERK activity are specifically sensitive to PERK inhibition; ectopic FOXO3 reduces this sensitivity.","method":"ChIP, siRNA knockdown, FOXO3 overexpression, Foxo1/3/4-/- MEF genetic validation, PERK inhibitor sensitivity assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-confirmed direct target, validated in triple-KO MEFs, functional drug sensitivity epistasis","pmids":["31312024"],"is_preprint":false},{"year":2021,"finding":"FOXO3a is transcriptionally activated by WTIP, which physically interacts with FOXO3a (Co-IP), promotes its nuclear translocation, and activates its downstream target PUMA, leading to intrinsic apoptosis in AML cells.","method":"Co-immunoprecipitation, nuclear translocation assays, luciferase reporter for FOXO3a transcriptional activity, PUMA expression assays, in vitro/in vivo apoptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional nuclear translocation and downstream target assays, single lab","pmids":["34930905"],"is_preprint":false},{"year":2022,"finding":"COPS3 enhances nuclear abundance of FOXO3 and expression of FOXO3-responsive autophagy genes to promote cisplatin resistance in osteosarcoma. In turn, FOXO3 inhibits ubiquitin-mediated degradation of COPS3 and attenuates SKP2-mediated COPS3 inhibition, forming a positive feedback loop. ChIP confirmed FOXO3 occupancy at autophagy gene promoters.","method":"ChIP, siRNA/shRNA knockdown, ubiquitination assays, cycloheximide chase, in vitro and xenograft cisplatin sensitivity assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and ubiquitination assays with functional rescue, single lab","pmids":["36451342"],"is_preprint":false},{"year":2022,"finding":"SIRT1 physically interacts with FOXO3 (Co-IP in RL95-2 cells) and deacetylates it. SIRT1-mediated FOXO3 deacetylation enhances FOXO3 activity, promoting BNIP3 transcription and PINK1/Parkin-mediated mitophagy, which drives hormone resistance in endometrial cancer. SIRT1 overexpression promotes cell proliferation, migration, and tumor growth.","method":"Co-immunoprecipitation, high-throughput transcriptome sequencing, FOXO3 acetylation assays, ChIP for BNIP3 promoter, PINK1/Parkin pathway assays, mouse tumor model","journal":"Molecular medicine (Cambridge, Mass.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ChIP plus functional pathway assays, single lab","pmids":["39266959"],"is_preprint":false},{"year":2022,"finding":"REP1 physically interacts with FOXO3 (identified by yeast two-hybrid and confirmed by Co-IP) and blocks FOXO3 nuclear translocation, thereby suppressing FOXO3-mediated apoptosis in colon cancer cells. REP1 silencing sensitizes cancer cells to serum starvation- and 5-FU-induced apoptosis; REP1 inhibition combined with 5-FU retards tumor growth in xenograft models.","method":"Yeast two-hybrid screen, Co-immunoprecipitation, nuclear translocation assays, siRNA knockdown, xenograft tumor model, apoptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Y2H plus reciprocal Co-IP plus functional localization and apoptosis assays, single lab","pmids":["28055019"],"is_preprint":false},{"year":2022,"finding":"FOXO3 transcriptionally activates PARKIN expression (demonstrated by ChIP and luciferase assays). FOXO3a-driven PARKIN upregulation restores mitophagy, thereby suppressing cardiac hypertrophy. Parkin transgenic mice subjected to Ang II show attenuated hypertrophy and improved cardiac function, dependent on FOXO3a-mediated PARKIN transcription.","method":"ChIP, luciferase reporter, PARKIN overexpression/knockdown, Parkin transgenic mice, Ang II cardiac hypertrophy model, echocardiography","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-confirmed transcriptional activation, transgenic mouse with functional cardiac readout, single lab","pmids":["36539848"],"is_preprint":false},{"year":2022,"finding":"FOXO3a transcriptional activation by SNAI2 is part of a coherent feed-forward loop: upon energy stress, FOXO3 transcriptionally induces SNAI2, and SNAI2 then interacts with FOXO3 to reinforce expression of autophagy genes. SNAI2 increases FOXO3-DNA binding, abrogating CRM1-dependent FOXO3 nuclear export and promoting nuclear retention of FOXO3. This loop is conserved in Drosophila (dFoxO-Snail).","method":"Genome-wide screen in HeLa cells, ChIP, Co-IP (SNAI2-FOXO3), nuclear export assays (CRM1 inhibition), Drosophila genetic epistasis, autophagy reporter assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide identification, ChIP, Co-IP, nuclear export mechanistic dissection, conserved in Drosophila","pmids":["35271390"],"is_preprint":false},{"year":2023,"finding":"FOXO3a directly binds to the SLC7A11 promoter to repress its expression, thereby reducing glutamate excitotoxicity during cerebral ischemia-reperfusion. AMPK activation leads to FOXO3a activation and inhibits mitochondria-associated ferroptosis. Loss of FoxO3a promotes mitochondrial hyperpolarization, oxygen consumption, and lipid peroxide accumulation.","method":"ChIP (FOXO3a on SLC7A11 promoter), FOXO3a KO/knockdown, AMPK inhibition, ferroptosis inducers (erastin), mitochondrial membrane potential assays, rat CIR injury model with trifluoperazine","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-confirmed promoter binding, FOXO3a KO with functional mitochondrial readouts, single lab","pmids":["37267686"],"is_preprint":false},{"year":2023,"finding":"FOXO3 directly binds the SPON1 promoter and selectively drives expression of the circular RNA form (circSPON1) but not SPON1 mRNA. circSPON1 then inhibits fibroblast activation by sequestering Smad3 in the cytoplasm and by sponging miR-942-5p/miR-520f-3p to promote Smad7 expression, thereby attenuating pulmonary fibrosis.","method":"ChIP (FOXO3 on SPON1 promoter), selective circRNA/mRNA expression analysis, Co-IP (circSPON1-Smad3), luciferase miRNA sponge assay, HFL1 fibroblast functional assays","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus Co-IP plus functional rescue, single lab","pmids":["37416778"],"is_preprint":false},{"year":2023,"finding":"METTL14 regulates FOXO3a expression and mRNA stability in a m6A-dependent manner. Loss of METTL14 decreases FOXO3a, impairing autophagic flux and aggravating inflammation in T cells from ankylosing spondylitis patients. Forced METTL14 expression upregulates FOXO3a, activates autophagy, and alleviates inflammation.","method":"m6A sequencing/meRIP, METTL14 knockdown/overexpression, FOXO3a mRNA stability assays, autophagy flux assays, T cell functional assays from patient samples","journal":"Clinical immunology (Orlando, Fla.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A-dependent mRNA regulation with mechanistic follow-up, single lab, two orthogonal methods","pmids":["37935312"],"is_preprint":false},{"year":2015,"finding":"FOXO3a mediates dexamethasone-induced cytotoxicity in B-ALL cells: FOXO3a translocates to the nucleus and induces p27Kip1 and Bim. Dexamethasone activates FOXO3a partly through PI3K/Akt suppression. Two specific post-translational modifications are associated with FOXO3a activation by dexamethasone: phosphorylation on Ser-7 (linked to p38/JNK) and acetylation on Lys-242/245 (correlated with SIRT1/2/6 downregulation and CBP/p300 induction).","method":"Immunoblot for phospho-Ser7 and acetyl-Lys242/245 FOXO3a, PI3K inhibitor, FOXO3a siRNA knockdown, nuclear translocation assays, p27Kip1 and Bim expression assays","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple PTM sites identified biochemically with functional nuclear translocation correlation, single lab","pmids":["26376801"],"is_preprint":false},{"year":2010,"finding":"FOXO3a directly activates SIRT6 transcription: ChIP and luciferase assays demonstrate FOXO3a enrichment at the SIRT6 promoter. The FOXO3a-SIRT6 axis suppresses aerobic glycolysis in melanoma, as SIRT6 knockdown rescues the anti-glycolytic effect of FOXO3a overexpression (replicated across two cancer types: melanoma and glioblastoma, separately confirmed).","method":"ChIP, luciferase reporter, knockdown/overexpression epistasis, glycolysis functional assays, mouse xenograft","journal":"International journal of oncology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP confirmed in two different cancer types (melanoma PMID:32124950; glioblastoma PMID:31004738), luciferase validation, epistatic rescue","pmids":["32124950","31004738"],"is_preprint":false}],"current_model":"FOXO3 (FKHRL1/FOXO3a) is a forkhead transcription factor whose activity is primarily controlled by phosphorylation-driven nuclear-cytoplasmic shuttling: Akt and SGK1 phosphorylate FOXO3a at Thr-32, Ser-253, and Ser-315 downstream of PI3K, causing nuclear export and transcriptional inactivation, while dephosphorylation allows nuclear accumulation and activation of target genes including pro-apoptotic Bim, Noxa, PUMA, and p27Kip1, the antioxidant MnSOD, and metabolic regulators such as SIRT6 (which suppresses the Warburg effect) and Keap1 (which constrains Nrf2); FOXO3a activity is further modulated by acetylation (by p300, reversed by SIRT1/2/6—promoting cytoplasmic relocalization and proteasomal degradation), by prolyl hydroxylation under normoxia (targeting it for proteasomal degradation), by interaction with ATM (which it activates via its FAT domain to coordinate DNA-damage response and apoptosis through the ATM-Chk2-p53 complex), and by a feed-forward nuclear-retention loop with SNAI2; in the nucleus FOXO3a directly transactivates Keap1, SIRT6, PERK, PARKIN, BNIP3, and SPON1 circRNA promoters while being transcriptionally regulated itself by TEAD1 and WTIP, collectively establishing FOXO3 as a central integrator of growth factor, metabolic, and stress signals that determines cell fate between survival, quiescence, autophagy, and apoptosis."},"narrative":{"mechanistic_narrative":"FOXO3 (FKHRL1/FOXO3a) is a forkhead transcription factor that integrates growth-factor, metabolic, and stress signals to determine cell fate between survival, quiescence, autophagy, and apoptosis [PMID:11694570, PMID:19896443]. Its activity is governed by phosphorylation-driven nuclear-cytoplasmic shuttling: downstream of PI3K, Akt phosphorylates Ser-253 and SGK1 phosphorylates Ser-315, with both kinases targeting Thr-32, and concerted phosphorylation of all three sites drives nuclear exclusion and transcriptional silencing [PMID:11154281]. This module is engaged by diverse upstream inputs including TGF-β, neurotrophin/TrkA signaling, and thrombopoietin, each acting through PI3K/Akt to retain FOXO3a in the cytoplasm [PMID:11694570, PMID:11953455, PMID:11278373], and by p53-induced SGK1 during DNA damage [PMID:15383658]. Layered onto phosphorylation, FOXO3a activity is tuned by acetylation—p300 acetylates FoxO3 to drive cytosolic relocalization and proteasomal degradation, while SIRT1 deacetylation enhances its activity—and by prolyl hydroxylation that targets it for degradation under normoxia [PMID:22094330, PMID:39266959, PMID:30912765]. When active in the nucleus, FOXO3a executes apoptosis through the mitochondrial pathway by transactivating the BH3-only proteins Bim and Noxa and repressing Survivin [PMID:16888645, PMID:19211844], and enforces cell-cycle arrest via p27Kip1 [PMID:17894357]. Beyond apoptosis, FOXO3a binds promoters of an antioxidant, metabolic, and autophagy program: it transactivates SIRT6 to suppress the Warburg effect [PMID:32124950, PMID:31004738], drives Keap1 transcription to constrain Nrf2 [PMID:26857210], activates BNIP3, PARKIN, and PERK to control mitophagy and the unfolded-protein response [PMID:27694219, PMID:36539848, PMID:31312024], and represses SLC7A11 to limit ferroptosis [PMID:37267686]. FOXO3 also coordinates the DNA-damage response by binding the ATM FAT domain and the ATM-Chk2-p53 complex to promote checkpoint activation, repair, and apoptosis [PMID:18344987, PMID:22893124]. Its own expression and localization are regulated by transcriptional activators (TEAD1, WTIP), m6A methylation (METTL14), nuclear-retention partners (SNAI2), and cytoplasmic-sequestering partners (REP1) [PMID:21211055, PMID:34930905, PMID:37935312, PMID:35271390, PMID:28055019], and FOXO3 maintains the neural stem cell pool by enforcing quiescence and controlling oxygen metabolism [PMID:19896443].","teleology":[{"year":2001,"claim":"Established the core regulatory logic of FOXO3a: how survival kinases inactivate it through site-specific phosphorylation.","evidence":"In vitro kinase assays with site-directed mutagenesis of Thr-32/Ser-253/Ser-315 and reporter/survival assays in cells","pmids":["11154281"],"confidence":"High","gaps":["Did not resolve which phosphatase reverses inactivation","In vivo physiological relevance of distinct Akt vs SGK1 site usage not established"]},{"year":2001,"claim":"Showed the phosphorylation switch is engaged by physiological extracellular cues, linking FOXO3a to TGF-β and thrombopoietin signaling and identifying its transactivation domain as required for apoptosis.","evidence":"Triple Akt-site mutant, dominant-negative Akt and PI3K inhibitor in mammary epithelial cells; inducible constitutively active allele in TPO-dependent megakaryocytic cells","pmids":["11694570","11278373"],"confidence":"High","gaps":["Direct target genes mediating arrest/apoptosis in these contexts not defined","Megakaryocyte study is Medium confidence with limited methods"]},{"year":2002,"claim":"Extended PI3K/Akt-mediated FOXO3a inactivation to neurotrophin-driven neuronal survival.","evidence":"Kinase inhibitors, constitutively active/dominant-negative Akt, in vitro kinase assay, TrkA vs p75 cell comparison in PC12 cells","pmids":["11953455"],"confidence":"Medium","gaps":["Downstream survival targets not identified","Single cell-line model"]},{"year":2004,"claim":"Revealed an Akt-independent route to FOXO3a inactivation during DNA damage, mediated by p53-induced SGK1.","evidence":"siRNA knockdown of SGK1, pharmacological inhibitors, localization and phosphorylation assays in p53-deficient cells","pmids":["15383658"],"confidence":"Medium","gaps":["Reconciliation with FOXO3a's pro-apoptotic role in DNA damage not fully resolved","Specific phosphosites engaged by p53/SGK1 not mapped"]},{"year":2006,"claim":"Defined the molecular effectors of FOXO3a-driven apoptosis, establishing it as a transcriptional trigger of the mitochondrial death pathway.","evidence":"4-OHT-regulated FKHRL1(A3)ER transgene, RNAi of Noxa/Bim, dnFADD, Bcl2 overexpression, cytochrome c and caspase assays; promoter binding to G6Pase IRSs","pmids":["16888645","16840535"],"confidence":"High","gaps":["Direct vs indirect transactivation of all BH3-only genes not fully dissected","Metabolic G6Pase study is Medium confidence"]},{"year":2008,"claim":"Identified a non-transcriptional, protein-protein function for FOXO3a in the DNA-damage response via direct ATM activation.","evidence":"Domain-mapped Co-IP (FOXO3a C-terminus to ATM FAT domain), siRNA, foci immunofluorescence, checkpoint and repair assays","pmids":["18344987"],"confidence":"High","gaps":["Structural basis of the FOXO3a-FAT interface not determined","How phosphorylation status of FOXO3a gates this interaction unclear"]},{"year":2008,"claim":"Resolved how FOXO3a enforces quiescence and survival in proliferating cells, distinguishing late vs early cell-cycle control and defining its stem-cell role.","evidence":"Adenoviral overexpression with p27Kip1/p21 promoter-luciferase in muscle precursors; FoxO3 knockout mice with neurosphere, multipotency, and expression profiling","pmids":["17894357","19896443"],"confidence":"High","gaps":["Direct FOXO3 occupancy of p27Kip1 promoter not shown by ChIP in muscle study (Medium)","NSC quiescence gene program members only partially defined"]},{"year":2009,"claim":"Showed FOXO3a sensitizes cells to genotoxic therapy by repressing the anti-apoptotic node Survivin.","evidence":"Conditional FKHRL1(A3)ER allele, Survivin shRNA and transgenic rescue, mitochondrial readouts, DNA-damaging drug sensitivity","pmids":["19211844"],"confidence":"High","gaps":["Direct binding of FOXO3a to BIRC5/Survivin regulatory regions not mapped","Generality across tumor types untested"]},{"year":2011,"claim":"Established acetylation by p300 as a degradation/relocalization switch and identified TEAD1 as an upstream transcriptional activator of FoxO3a in muscle.","evidence":"Co-IP (FoxO3-p300), acetylation-mimic/resistant mutants (Lys-262) in denervation model; ChIP/EMSA/luciferase for TEAD1 at the FoxO3a M-CAT element","pmids":["22094330","21211055"],"confidence":"High","gaps":["Deacetylases acting on these lysines in muscle not identified in this study","TEAD1 study is Medium confidence and muscle-restricted"]},{"year":2012,"claim":"Placed FOXO3 inside the ATM-Chk2-p53 DNA-damage complex as an essential bidirectional partner and defined a FOXO3-ATM-CREB axis controlling caspase-8.","evidence":"Reciprocal Co-IP of the four-protein complex, siRNA epistasis, foci and apoptosis assays; conditional FOXO3 transgene with ATM/CREB inhibition and bisulfite methylation analysis","pmids":["22893124","22493319"],"confidence":"High","gaps":["Stoichiometry and assembly order of the FOXO3-ATM-Chk2-p53 complex unresolved","caspase-8 axis study is Medium confidence"]},{"year":2016,"claim":"Defined FOXO3 as a transcriptional governor of redox balance through direct Keap1 activation and BNIP3-driven mitochondrial fate.","evidence":"ChIP, siRNA, luciferase and xenograft for the FoxO3-Keap1-Nrf2 axis; dominant-negative FOXO3a (AAV9) with BNIP3 manipulation and cardiac functional readouts","pmids":["26857210","27694219"],"confidence":"High","gaps":["Interplay between FOXO3-driven Keap1 (anti-Nrf2) and FOXO3's own antioxidant role not integrated","Tissue-specific weighting of these outputs unclear"]},{"year":2019,"claim":"Identified prolyl hydroxylation as an oxygen-sensing degradation switch and established direct FOXO3 transactivation of metabolic/stress targets SIRT6 and PERK.","evidence":"Tubule-specific FoxO3 and HIF-1α conditional KO with prolyl hydroxylation assays and renal injury models; ChIP/luciferase/triple-KO MEF validation for SIRT6 and PERK targets; NMR-validated small molecules targeting the FOXO3 DNA-binding domain","pmids":["30912765","31004738","32124950","31312024","31789593"],"confidence":"High","gaps":["The PHD enzyme(s) and hydroxylated proline residues not all individually mapped","Druggability shown in cells but not in vivo efficacy"]},{"year":2010,"claim":"Confirmed direct FOXO3a transactivation of SIRT6 to suppress aerobic glycolysis across multiple cancer types.","evidence":"ChIP, luciferase, knockdown/overexpression epistasis with glycolysis assays and xenografts in melanoma and glioblastoma","pmids":["32124950","31004738"],"confidence":"High","gaps":["Whether the FOXO3-SIRT6 axis operates in non-transformed metabolic tissues untested"]},{"year":2022,"claim":"Expanded FOXO3's transcriptional output to autophagy/mitophagy and revealed positive feedback loops and partner-controlled localization that amplify or constrain its activity.","evidence":"ChIP and feedback dissection for COPS3 and the SNAI2-FOXO3 feed-forward loop (with CRM1 export assays, conserved in Drosophila); Co-IP for SIRT1 (BNIP3/PINK1-Parkin) and REP1 (cytoplasmic sequestration); ChIP/luciferase for PARKIN with transgenic cardiac rescue","pmids":["36451342","35271390","39266959","28055019","36539848"],"confidence":"High","gaps":["Several partner interactions rest on single-lab Co-IP (Medium)","Hierarchy among competing nuclear-retention and export factors unresolved"]},{"year":2023,"claim":"Showed FOXO3a controls non-canonical outputs—ferroptosis suppression via SLC7A11 repression, selective circRNA biogenesis (circSPON1), and its own m6A-dependent stability.","evidence":"ChIP and FOXO3a KO with AMPK/ferroptosis readouts (SLC7A11); ChIP plus selective circRNA expression and Co-IP (circSPON1-Smad3); m6A-seq/meRIP and mRNA stability assays (METTL14)","pmids":["37267686","37416778","37935312"],"confidence":"Medium","gaps":["Mechanism of FOXO3 selectivity for circRNA over mRNA at SPON1 not defined","All three findings are single-lab Medium-confidence"]},{"year":null,"claim":"How the many competing post-translational modifications, partner interactions, and feedback loops are integrated to set FOXO3 nuclear residence and target-gene selection in a given cell state remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No quantitative model linking PTM combinations to specific target-gene programs","Structural basis for FOXO3 partner discrimination unknown","Mechanism of promoter selectivity (apoptosis vs autophagy vs metabolic targets) undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,5,16,19,22,28,29,33]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,13,16,19,21,29,30,33]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,8,12,23,26,28]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,12,26]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[14,16,19]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,10,14,23]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[16,19,22,27,33]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8,14]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[24,25,28,31]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,9,11]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[16,18,29]}],"complexes":["ATM-Chk2-p53 complex"],"partners":["ATM","AKT1","SGK1","EP300","SIRT1","SNAI2","WTIP","REP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43524","full_name":"Forkhead box protein O3","aliases":["AF6q21 protein","Forkhead in rhabdomyosarcoma-like 1"],"length_aa":673,"mass_kda":71.3,"function":"Transcriptional activator that recognizes and binds to the DNA sequence 5'-[AG]TAAA[TC]A-3' and regulates different processes, such as apoptosis and autophagy (PubMed:10102273, PubMed:16751106, PubMed:21329882, PubMed:30513302). Acts as a positive regulator of autophagy in skeletal muscle: in starved cells, enters the nucleus following dephosphorylation and binds the promoters of autophagy genes, such as GABARAP1L, MAP1LC3B and ATG12, thereby activating their expression, resulting in proteolysis of skeletal muscle proteins (By similarity). Triggers apoptosis in the absence of survival factors, including neuronal cell death upon oxidative stress (PubMed:10102273, PubMed:16751106). Participates in post-transcriptional regulation of MYC: following phosphorylation by MAPKAPK5, promotes induction of miR-34b and miR-34c expression, 2 post-transcriptional regulators of MYC that bind to the 3'UTR of MYC transcript and prevent its translation (PubMed:21329882). In response to metabolic stress, translocates into the mitochondria where it promotes mtDNA transcription (PubMed:23283301). In response to metabolic stress, translocates into the mitochondria where it promotes mtDNA transcription. Also acts as a key regulator of chondrogenic commitment of skeletal progenitor cells in response to lipid availability: when lipids levels are low, translocates to the nucleus and promotes expression of SOX9, which induces chondrogenic commitment and suppresses fatty acid oxidation (By similarity). Also acts as a key regulator of regulatory T-cells (Treg) differentiation by activating expression of FOXP3 (PubMed:30513302)","subcellular_location":"Cytoplasm, cytosol; Nucleus; Mitochondrion matrix; Mitochondrion outer membrane","url":"https://www.uniprot.org/uniprotkb/O43524/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FOXO3","classification":"Not Classified","n_dependent_lines":269,"n_total_lines":1090,"dependency_fraction":0.2467889908256881},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FOXO3","total_profiled":1310},"omim":[{"mim_id":"621322","title":"FOXO-INDUCED LONG NONCODING RNA 1; FILNC1","url":"https://www.omim.org/entry/621322"},{"mim_id":"619624","title":"LEMUR TYROSINE KINASE 3; LMTK3","url":"https://www.omim.org/entry/619624"},{"mim_id":"616186","title":"H19/IGF2-IMPRINTING CONTROL REGION","url":"https://www.omim.org/entry/616186"},{"mim_id":"611607","title":"MICRO RNA 182; MIR182","url":"https://www.omim.org/entry/611607"},{"mim_id":"611457","title":"FORKHEAD BOX O6; FOXO6","url":"https://www.omim.org/entry/611457"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FOXO3"},"hgnc":{"alias_symbol":["AF6q21","FOXO2"],"prev_symbol":["FKHRL1","FOXO3A"]},"alphafold":{"accession":"O43524","domains":[{"cath_id":"1.10.10.10","chopping":"163-237","consensus_level":"high","plddt":90.8583,"start":163,"end":237}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43524","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43524-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43524-F1-predicted_aligned_error_v6.png","plddt_mean":50.66},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FOXO3","jax_strain_url":"https://www.jax.org/strain/search?query=FOXO3"},"sequence":{"accession":"O43524","fasta_url":"https://rest.uniprot.org/uniprotkb/O43524.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43524/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43524"}},"corpus_meta":[{"pmid":"26861625","id":"PMC_26861625","title":"Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2.","date":"2016","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/26861625","citation_count":1370,"is_preprint":false},{"pmid":"11154281","id":"PMC_11154281","title":"Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a).","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11154281","citation_count":727,"is_preprint":false},{"pmid":"19896443","id":"PMC_19896443","title":"FoxO3 regulates neural stem cell homeostasis.","date":"2009","source":"Cell stem cell","url":"https://pubmed.ncbi.nlm.nih.gov/19896443","citation_count":476,"is_preprint":false},{"pmid":"30045773","id":"PMC_30045773","title":"Critical role of FOXO3a in carcinogenesis.","date":"2018","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30045773","citation_count":390,"is_preprint":false},{"pmid":"25832544","id":"PMC_25832544","title":"FOXO3: A Major Gene for Human Longevity--A Mini-Review.","date":"2015","source":"Gerontology","url":"https://pubmed.ncbi.nlm.nih.gov/25832544","citation_count":277,"is_preprint":false},{"pmid":"25199915","id":"PMC_25199915","title":"GWAS of longevity in CHARGE consortium confirms APOE and FOXO3 candidacy.","date":"2014","source":"The journals of gerontology. 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Phosphorylation of all three sites by SGK1 and Akt in concert suppresses FOXO3a-dependent transcription, leading to nuclear exclusion and inhibition of cell cycle arrest and apoptosis. SGK1 acts downstream of PI3K, like Akt, and promotes cell survival in part through this mechanism.\",\n      \"method\": \"In vitro kinase assays, site-directed mutagenesis of phosphorylation sites, overexpression and dominant-negative constructs, transcriptional reporter assays, cell survival assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis at three defined sites, multiple orthogonal methods, replicated across conditions\",\n      \"pmids\": [\"11154281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TGF-β induces phosphorylation and cytoplasmic retention of FOXO3a (FKHRL1) in mammary epithelial cells via PI3K/Akt. A triple Akt-site mutant of FKHRL1 (TM-FKHRL1) that cannot be phosphorylated by Akt fails to translocate to the cytoplasm in response to TGF-β, and dominant-negative Akt blocks TGF-β-induced FKHRL1 nuclear exclusion, phosphorylation, and transcriptional suppression. FKHRL1-driven apoptosis requires its transactivation domain.\",\n      \"method\": \"Triple phosphosite mutant transfection, dominant-negative Akt, PI3K inhibitor (LY294002), nuclear exclusion assays, transcriptional reporter assays, apoptosis assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of all three Akt sites, dominant-negative Akt, PI3K inhibitor, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"11694570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"FOXO3a (FKHRL1) is expressed in human megakaryocytes and is phosphorylated by thrombopoietin (TPO) downstream of PI3K/Akt. Induced expression of constitutively active FKHRL1 causes G0/G1 cell cycle arrest in TPO-dependent megakaryocytic cells, identifying FKHRL1 as a downstream regulator of cell cycle in megakaryocytopoiesis.\",\n      \"method\": \"Tetracycline-inducible expression system, cell cycle analysis (flow cytometry), PI3K/Akt phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible expression system with defined cell cycle readout, single lab, two methods\",\n      \"pmids\": [\"11278373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NGF rapidly induces phosphorylation of FKHRL1 (FOXO3a) in PC12 cells via TrkA receptor and PI3K/Akt kinase (but not MAP kinase or S6p70 kinase). Constitutively active Akt and in vitro kinase assays confirmed Akt as the mediating kinase. This phosphorylation contributes to neurotrophin-mediated cell survival.\",\n      \"method\": \"Kinase inhibitors, constitutively active and dominant-negative Akt transfection, in vitro kinase assay, TrkA-expressing vs p75-expressing cell comparison, survival assays with PI3K inhibitor\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus multiple genetic tools, single lab\",\n      \"pmids\": [\"11953455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In response to DNA damage, p53 activation leads to FOXO3a (FKHRL1) phosphorylation and cytoplasmic translocation via SGK1, which is induced by p53 in an ERK1/2-dependent post-translational manner. AKT is dispensable for this p53-dependent suppression of FKHRL1; siRNA knockdown of SGK1 significantly decreases FKHRL1 phosphorylation after DNA damage.\",\n      \"method\": \"siRNA knockdown of SGK1, pharmacological inhibitors, subcellular localization assays, Western blotting for phosphorylation, genetic analysis in p53-deficient cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown combined with pharmacological inhibitors and localization assays, single lab\",\n      \"pmids\": [\"15383658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"FOXO3a (FKHRL1) induces apoptosis in neuroblastoma cells through the mitochondrial pathway by transcriptionally activating the BH3-only proteins Noxa and Bim, which together are critical for cytochrome c release and caspase-dependent apoptosis. RNAi knockdown of either Noxa or Bim reduces FOXO3a-induced cell death. The death-receptor pathway (TRAIL/dnFADD) is not required, whereas Bcl2 overexpression protects, confirming the Bcl2-rheostat model.\",\n      \"method\": \"4-OH-tamoxifen-regulated FKHRL1(A3)ERtm transgene, RNAi knockdown of Noxa and Bim, dominant-negative FADD expression, Bcl2 overexpression, cytochrome c release assay, caspase activity\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional transgene, RNAi of specific targets, multiple orthogonal death assays, replicated with multiple genetic perturbations\",\n      \"pmids\": [\"16888645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"FOXO3a (FKHRL1) and FOXO1a bind insulin response sequences (IRSs) in the glucose-6-phosphatase (G6Pase) promoter cooperatively (two adjacent IRSs), and their binding correlates with insulin-mediated inhibition of basal G6Pase transcription. Insulin stimulates nuclear exclusion of FOXO1a/3a in a PI3K-dependent manner in granulosa cells.\",\n      \"method\": \"Gel retardation/binding assays, overexpression experiments, fusion gene transcription assays in multiple promoter contexts, PI3K inhibition\",\n      \"journal\": \"Molecular endocrinology (Baltimore, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct DNA binding assays with overexpression and promoter constructs, single lab, two orthogonal methods\",\n      \"pmids\": [\"16840535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"AGE-induced oxidative stress causes phosphorylation of FOXO3a (FKHRL1) in a redox-dependent and p66shc Ser-36-phosphorylation-dependent manner, leading to ~70% downregulation of MnSOD. AGER1 overexpression suppresses these pro-oxidant responses; AGER1 siRNA restores them. PI3K inhibitor (LY-294002) and N-acetylcysteine partially block FKHRL1 phosphorylation-driven MnSOD suppression.\",\n      \"method\": \"p66shc Ser-36 mutants, AGER1 overexpression/siRNA, PI3K inhibitor, antioxidant rescue, Western blotting\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined mutants and siRNA, single lab, multiple inhibitors\",\n      \"pmids\": [\"18032526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FOXO3a interacts with the ATM FAT domain (C-terminal domain of FOXO3a binds FAT domain of ATM) and promotes ATM autophosphorylation at Ser-1981, leading to formation of ATM-pS1981 and γ-H2AX nuclear foci in response to DNA damage. Silencing FOXO3a abrogates ATM foci formation and DNA repair. FOXO3a promotes intra-S and G2/M checkpoints and facilitates DNA repair.\",\n      \"method\": \"Co-immunoprecipitation, domain-mapping, siRNA knockdown, immunofluorescence foci assays, cell cycle checkpoint analysis, DNA repair assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-mapped Co-IP, siRNA loss-of-function, multiple functional readouts (foci, checkpoints, repair), single rigorous study\",\n      \"pmids\": [\"18344987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FoxO3a overexpression in muscle precursor cells (MPCs) impairs proliferation by preferentially inducing p27Kip1 promoter activity and protein expression (but not p21Waf/Cip1, which is decreased). FoxO3a overexpression does not affect retinoblastoma phosphorylation or cyclin D1, indicating it does not impair the early G1/S transition.\",\n      \"method\": \"Adenoviral FoxO3a overexpression, BrdU incorporation assay, promoter-luciferase assays for p27Kip1 and p21Waf/Cip1, Western blotting\",\n      \"journal\": \"Muscle & nerve\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression with promoter-luciferase and protein readouts, single lab\",\n      \"pmids\": [\"17894357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"FOXO3a (FKHRL1) represses BIRC5/Survivin transcription and protein expression. Conditional FKHRL1 activation sensitizes neuroblastoma cells to DNA-damaging agents (doxorubicin, etoposide). Transgenic Survivin rescues FKHRL1-induced apoptosis and prevents Bim/Bax mitochondrial accumulation, cytochrome c release, and loss of mitochondrial membrane potential. Survivin shRNA accelerates FKHRL1-induced apoptosis.\",\n      \"method\": \"4-OH-tamoxifen-regulated FKHRL1(A3)ERtm allele, retroviral Survivin shRNA, transgenic Survivin rescue, cytochrome c release, mitochondrial membrane potential assay, drug sensitivity assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional transgene, shRNA knockdown, transgenic rescue, multiple orthogonal mitochondrial readouts\",\n      \"pmids\": [\"19211844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"FoxO3 is required for neural stem cell (NSC) pool maintenance. FoxO3-/- adult mice have fewer NSCs in vivo, and NSCs from FoxO3-/- mice show decreased self-renewal and impaired multipotency. FoxO3 regulates a gene expression program in NSCs that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism.\",\n      \"method\": \"FoxO3 knockout mice, neurosphere assays (self-renewal), multipotency assays, gene expression profiling\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with in vivo and in vitro cellular phenotype, gene expression profiling, multiple orthogonal readouts\",\n      \"pmids\": [\"19896443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"During denervation-induced muscle atrophy, FoxO3 is progressively acetylated by the histone acetyltransferase p300 (demonstrated by co-immunoprecipitation). Acetylation causes cytosolic relocalization and proteasomal degradation of FoxO3, coinciding with downregulation of its target atrogin-1. Acetylation-mimicking FoxO3 mutants show decreased transcriptional activity and cytosolic localization; Lys-262 is critical for FoxO3 translocation. Acetylation thus negatively modulates FoxO3 activity.\",\n      \"method\": \"Co-immunoprecipitation (FoxO3-p300), acetylation-mimic and acetylation-resistant FoxO3 mutants, denervation mouse model, subcellular fractionation, proteasome inhibition\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP with defined acetyltransferase, site-directed mutagenesis of multiple lysines, in vivo denervation model, proteasome inhibition rescue\",\n      \"pmids\": [\"22094330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TEAD1 directly binds the M-CAT element in the FoxO3a promoter and transcriptionally activates FoxO3a expression in skeletal muscle, as demonstrated by ChIP, EMSA, and luciferase reporter assays. Overexpression and inhibition experiments confirm TEAD1 as a positive regulator of FoxO3a.\",\n      \"method\": \"ChIP-on-chip, independent ChIP-PCR, EMSA, luciferase reporter assays, overexpression and inhibition experiments\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal DNA-binding methods (ChIP, EMSA, luciferase), single lab\",\n      \"pmids\": [\"21211055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"FOXO3 interacts with the ATM-Chk2-p53 complex (demonstrated by Co-IP), augments phosphorylation of all complex members, and induces nuclear foci formation upon DNA damage. FOXO3 is essential for DNA damage-induced apoptosis; conversely, FOXO3 requires ATM, Chk2, and phospho-p53 isoforms to trigger apoptosis. FOXO3 also contributes to chromatin retention of phosphorylated p53.\",\n      \"method\": \"Co-immunoprecipitation of FOXO3-ATM-Chk2-p53 complex, siRNA knockdown, nuclear foci immunofluorescence, apoptosis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP of multi-protein complex, genetic epistasis by siRNA of multiple pathway members, multiple functional readouts\",\n      \"pmids\": [\"22893124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Conditional FOXO3 activation induces caspase-8 expression in neuroblastoma without changing DNA methylation of the caspase-8 gene. Instead, FOXO3 induces phosphorylation of ATM, which then phosphorylates CREB; CREB phosphorylation is critical for FOXO3-mediated caspase-8 expression. The DNA-demethylating drug 5-azadC activates this FOXO3-ATM-CREB pathway to restore caspase-8 expression.\",\n      \"method\": \"Conditional FOXO3 transgene (ERtm), ATM inhibitor/knockdown, CREB inhibition, DNA methylation analysis (bisulfite), caspase-8 expression assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional transgene with mechanistic dissection using ATM and CREB inhibitors, single lab\",\n      \"pmids\": [\"22493319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FOXO3a directly binds to the Keap1 promoter and drives basal Keap1 transcription. FoxO3 depletion reduces Keap1, thereby stabilizing and activating Nrf2. This FoxO3-Keap1-Nrf2 axis explains how AKT activation or TNF-α-mediated FoxO3 inactivation leads to Nrf2 induction. In vivo, FoxO3 deficiency potentiates tumor formation and cisplatin resistance in cholangiocarcinoma through Nrf2 activation.\",\n      \"method\": \"ChIP assay (FoxO3 on Keap1 promoter), siRNA knockdown, luciferase reporter, xenograft tumor model, ROS measurement\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-confirmed promoter binding, siRNA KD with multiple epistasis experiments, in vivo xenograft model\",\n      \"pmids\": [\"26857210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FOXO3a transcriptionally regulates BNIP3 expression in cardiomyocytes. Increased FOXO3a activity upregulates BNIP3, leading to increased mitochondrial Ca2+, decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis in adult cardiomyocytes. Dominant-negative FOXO3a (AAV9) in a rat HFpEF model decreases BNIP3, reverses adverse remodeling, and improves cardiac function.\",\n      \"method\": \"Dominant-negative FOXO3a adenovirus and AAV9 in vitro/in vivo, BNIP3 knockdown/overexpression, mitochondrial membrane potential assay, Ca2+ imaging, cardiac functional analysis (echocardiography)\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative construct in vitro and in vivo AAV delivery, multiple mitochondrial readouts, disease model rescue\",\n      \"pmids\": [\"27694219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FoxO3 is regulated by prolyl hydroxylation: hypoxia inhibits prolyl hydroxylase (PHD)-mediated prolyl hydroxylation of FoxO3, preventing its degradation and leading to FoxO3 accumulation in renal tubular cells. Hypoxia-activated HIF-1α contributes to FoxO3 activation; tubular HIF-1α deletion reduces FoxO3 activation. Tubular FoxO3 deletion during AKI-to-CKD transition worsens structural/functional damage, decreases autophagy, and increases oxidative injury.\",\n      \"method\": \"Tubule-specific conditional FoxO3 knockout and HIF-1α knockout mice, prolyl hydroxylation assays, renal ischemia/reperfusion model, autophagy and oxidative stress markers\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO of FoxO3 and HIF-1α in same tissue, biochemical prolyl hydroxylation evidence, disease model with functional readouts\",\n      \"pmids\": [\"30912765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FOXO3 directly transactivates SIRT6 by binding to its promoter region (confirmed by luciferase assay and ChIP). The FOXO3a-SIRT6 axis suppresses aerobic glycolysis (Warburg effect) in melanoma cells. SIRT6 knockdown or overexpression rescues the glycolytic effects of FOXO3a manipulation.\",\n      \"method\": \"ChIP assay, luciferase reporter assay, lentiviral SIRT6 knockdown/overexpression rescue, glycolysis assays (glucose uptake, lactate, Seahorse), mouse xenograft\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP and luciferase confirm direct promoter binding, epistatic rescue experiments with multiple metabolic readouts\",\n      \"pmids\": [\"32124950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FOXO3 directly binds the SIRT6 promoter in glioblastoma cells and transcriptionally activates SIRT6, thereby suppressing glycolysis (Warburg effect). FKHRL1 knockdown reduces SIRT6 expression and increases glycolysis; SIRT6 restoration reverses the glycolytic phenotype caused by FKHRL1 knockdown. This axis is confirmed both in vitro and in vivo.\",\n      \"method\": \"Luciferase assay, ChIP assay, siRNA knockdown, SIRT6 overexpression rescue, Seahorse glycolysis assay, xenograft model\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP and luciferase assays confirm direct binding; epistatic rescue experiments; in vivo validation\",\n      \"pmids\": [\"31004738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Small molecules targeting the FOXO3 DNA-binding domain (DBD) physically interact with it (validated by NMR spectroscopy and docking). Compounds S9 and S9OX interfere with FOXO3 target promoter binding, gene transcription, and modulate the physiological transcriptional program in cancer cells, demonstrating the druggability of the FOXO3-DBD.\",\n      \"method\": \"Pharmacophore-modeling in silico screen, fluorescence polarization binding assay, NMR spectroscopy, docking studies, transcriptional target assays in human cells\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural validation of binding, in vitro fluorescence polarization assay, functional transcriptional readout, single lab\",\n      \"pmids\": [\"31789593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FOXO3 directly transcribes PERK (eIF2AK3) as established by ChIP, siRNA knockdown, and overexpression assays, as well as validation in Foxo1/3/4-/- MEFs. Drug-resistant breast cancer cells with low FOXO3/PERK expression and high PERK activity are specifically sensitive to PERK inhibition; ectopic FOXO3 reduces this sensitivity.\",\n      \"method\": \"ChIP, siRNA knockdown, FOXO3 overexpression, Foxo1/3/4-/- MEF genetic validation, PERK inhibitor sensitivity assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-confirmed direct target, validated in triple-KO MEFs, functional drug sensitivity epistasis\",\n      \"pmids\": [\"31312024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FOXO3a is transcriptionally activated by WTIP, which physically interacts with FOXO3a (Co-IP), promotes its nuclear translocation, and activates its downstream target PUMA, leading to intrinsic apoptosis in AML cells.\",\n      \"method\": \"Co-immunoprecipitation, nuclear translocation assays, luciferase reporter for FOXO3a transcriptional activity, PUMA expression assays, in vitro/in vivo apoptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional nuclear translocation and downstream target assays, single lab\",\n      \"pmids\": [\"34930905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"COPS3 enhances nuclear abundance of FOXO3 and expression of FOXO3-responsive autophagy genes to promote cisplatin resistance in osteosarcoma. In turn, FOXO3 inhibits ubiquitin-mediated degradation of COPS3 and attenuates SKP2-mediated COPS3 inhibition, forming a positive feedback loop. ChIP confirmed FOXO3 occupancy at autophagy gene promoters.\",\n      \"method\": \"ChIP, siRNA/shRNA knockdown, ubiquitination assays, cycloheximide chase, in vitro and xenograft cisplatin sensitivity assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and ubiquitination assays with functional rescue, single lab\",\n      \"pmids\": [\"36451342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIRT1 physically interacts with FOXO3 (Co-IP in RL95-2 cells) and deacetylates it. SIRT1-mediated FOXO3 deacetylation enhances FOXO3 activity, promoting BNIP3 transcription and PINK1/Parkin-mediated mitophagy, which drives hormone resistance in endometrial cancer. SIRT1 overexpression promotes cell proliferation, migration, and tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, high-throughput transcriptome sequencing, FOXO3 acetylation assays, ChIP for BNIP3 promoter, PINK1/Parkin pathway assays, mouse tumor model\",\n      \"journal\": \"Molecular medicine (Cambridge, Mass.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ChIP plus functional pathway assays, single lab\",\n      \"pmids\": [\"39266959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"REP1 physically interacts with FOXO3 (identified by yeast two-hybrid and confirmed by Co-IP) and blocks FOXO3 nuclear translocation, thereby suppressing FOXO3-mediated apoptosis in colon cancer cells. REP1 silencing sensitizes cancer cells to serum starvation- and 5-FU-induced apoptosis; REP1 inhibition combined with 5-FU retards tumor growth in xenograft models.\",\n      \"method\": \"Yeast two-hybrid screen, Co-immunoprecipitation, nuclear translocation assays, siRNA knockdown, xenograft tumor model, apoptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Y2H plus reciprocal Co-IP plus functional localization and apoptosis assays, single lab\",\n      \"pmids\": [\"28055019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FOXO3 transcriptionally activates PARKIN expression (demonstrated by ChIP and luciferase assays). FOXO3a-driven PARKIN upregulation restores mitophagy, thereby suppressing cardiac hypertrophy. Parkin transgenic mice subjected to Ang II show attenuated hypertrophy and improved cardiac function, dependent on FOXO3a-mediated PARKIN transcription.\",\n      \"method\": \"ChIP, luciferase reporter, PARKIN overexpression/knockdown, Parkin transgenic mice, Ang II cardiac hypertrophy model, echocardiography\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-confirmed transcriptional activation, transgenic mouse with functional cardiac readout, single lab\",\n      \"pmids\": [\"36539848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FOXO3a transcriptional activation by SNAI2 is part of a coherent feed-forward loop: upon energy stress, FOXO3 transcriptionally induces SNAI2, and SNAI2 then interacts with FOXO3 to reinforce expression of autophagy genes. SNAI2 increases FOXO3-DNA binding, abrogating CRM1-dependent FOXO3 nuclear export and promoting nuclear retention of FOXO3. This loop is conserved in Drosophila (dFoxO-Snail).\",\n      \"method\": \"Genome-wide screen in HeLa cells, ChIP, Co-IP (SNAI2-FOXO3), nuclear export assays (CRM1 inhibition), Drosophila genetic epistasis, autophagy reporter assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide identification, ChIP, Co-IP, nuclear export mechanistic dissection, conserved in Drosophila\",\n      \"pmids\": [\"35271390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FOXO3a directly binds to the SLC7A11 promoter to repress its expression, thereby reducing glutamate excitotoxicity during cerebral ischemia-reperfusion. AMPK activation leads to FOXO3a activation and inhibits mitochondria-associated ferroptosis. Loss of FoxO3a promotes mitochondrial hyperpolarization, oxygen consumption, and lipid peroxide accumulation.\",\n      \"method\": \"ChIP (FOXO3a on SLC7A11 promoter), FOXO3a KO/knockdown, AMPK inhibition, ferroptosis inducers (erastin), mitochondrial membrane potential assays, rat CIR injury model with trifluoperazine\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-confirmed promoter binding, FOXO3a KO with functional mitochondrial readouts, single lab\",\n      \"pmids\": [\"37267686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FOXO3 directly binds the SPON1 promoter and selectively drives expression of the circular RNA form (circSPON1) but not SPON1 mRNA. circSPON1 then inhibits fibroblast activation by sequestering Smad3 in the cytoplasm and by sponging miR-942-5p/miR-520f-3p to promote Smad7 expression, thereby attenuating pulmonary fibrosis.\",\n      \"method\": \"ChIP (FOXO3 on SPON1 promoter), selective circRNA/mRNA expression analysis, Co-IP (circSPON1-Smad3), luciferase miRNA sponge assay, HFL1 fibroblast functional assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus Co-IP plus functional rescue, single lab\",\n      \"pmids\": [\"37416778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"METTL14 regulates FOXO3a expression and mRNA stability in a m6A-dependent manner. Loss of METTL14 decreases FOXO3a, impairing autophagic flux and aggravating inflammation in T cells from ankylosing spondylitis patients. Forced METTL14 expression upregulates FOXO3a, activates autophagy, and alleviates inflammation.\",\n      \"method\": \"m6A sequencing/meRIP, METTL14 knockdown/overexpression, FOXO3a mRNA stability assays, autophagy flux assays, T cell functional assays from patient samples\",\n      \"journal\": \"Clinical immunology (Orlando, Fla.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A-dependent mRNA regulation with mechanistic follow-up, single lab, two orthogonal methods\",\n      \"pmids\": [\"37935312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FOXO3a mediates dexamethasone-induced cytotoxicity in B-ALL cells: FOXO3a translocates to the nucleus and induces p27Kip1 and Bim. Dexamethasone activates FOXO3a partly through PI3K/Akt suppression. Two specific post-translational modifications are associated with FOXO3a activation by dexamethasone: phosphorylation on Ser-7 (linked to p38/JNK) and acetylation on Lys-242/245 (correlated with SIRT1/2/6 downregulation and CBP/p300 induction).\",\n      \"method\": \"Immunoblot for phospho-Ser7 and acetyl-Lys242/245 FOXO3a, PI3K inhibitor, FOXO3a siRNA knockdown, nuclear translocation assays, p27Kip1 and Bim expression assays\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple PTM sites identified biochemically with functional nuclear translocation correlation, single lab\",\n      \"pmids\": [\"26376801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"FOXO3a directly activates SIRT6 transcription: ChIP and luciferase assays demonstrate FOXO3a enrichment at the SIRT6 promoter. The FOXO3a-SIRT6 axis suppresses aerobic glycolysis in melanoma, as SIRT6 knockdown rescues the anti-glycolytic effect of FOXO3a overexpression (replicated across two cancer types: melanoma and glioblastoma, separately confirmed).\",\n      \"method\": \"ChIP, luciferase reporter, knockdown/overexpression epistasis, glycolysis functional assays, mouse xenograft\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP confirmed in two different cancer types (melanoma PMID:32124950; glioblastoma PMID:31004738), luciferase validation, epistatic rescue\",\n      \"pmids\": [\"32124950\", \"31004738\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FOXO3 (FKHRL1/FOXO3a) is a forkhead transcription factor whose activity is primarily controlled by phosphorylation-driven nuclear-cytoplasmic shuttling: Akt and SGK1 phosphorylate FOXO3a at Thr-32, Ser-253, and Ser-315 downstream of PI3K, causing nuclear export and transcriptional inactivation, while dephosphorylation allows nuclear accumulation and activation of target genes including pro-apoptotic Bim, Noxa, PUMA, and p27Kip1, the antioxidant MnSOD, and metabolic regulators such as SIRT6 (which suppresses the Warburg effect) and Keap1 (which constrains Nrf2); FOXO3a activity is further modulated by acetylation (by p300, reversed by SIRT1/2/6—promoting cytoplasmic relocalization and proteasomal degradation), by prolyl hydroxylation under normoxia (targeting it for proteasomal degradation), by interaction with ATM (which it activates via its FAT domain to coordinate DNA-damage response and apoptosis through the ATM-Chk2-p53 complex), and by a feed-forward nuclear-retention loop with SNAI2; in the nucleus FOXO3a directly transactivates Keap1, SIRT6, PERK, PARKIN, BNIP3, and SPON1 circRNA promoters while being transcriptionally regulated itself by TEAD1 and WTIP, collectively establishing FOXO3 as a central integrator of growth factor, metabolic, and stress signals that determines cell fate between survival, quiescence, autophagy, and apoptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FOXO3 (FKHRL1/FOXO3a) is a forkhead transcription factor that integrates growth-factor, metabolic, and stress signals to determine cell fate between survival, quiescence, autophagy, and apoptosis [#1, #11]. Its activity is governed by phosphorylation-driven nuclear-cytoplasmic shuttling: downstream of PI3K, Akt phosphorylates Ser-253 and SGK1 phosphorylates Ser-315, with both kinases targeting Thr-32, and concerted phosphorylation of all three sites drives nuclear exclusion and transcriptional silencing [#0]. This module is engaged by diverse upstream inputs including TGF-\\u03b2, neurotrophin/TrkA signaling, and thrombopoietin, each acting through PI3K/Akt to retain FOXO3a in the cytoplasm [#1, #3, #2], and by p53-induced SGK1 during DNA damage [#4]. Layered onto phosphorylation, FOXO3a activity is tuned by acetylation\\u2014p300 acetylates FoxO3 to drive cytosolic relocalization and proteasomal degradation, while SIRT1 deacetylation enhances its activity\\u2014and by prolyl hydroxylation that targets it for degradation under normoxia [#12, #25, #18]. When active in the nucleus, FOXO3a executes apoptosis through the mitochondrial pathway by transactivating the BH3-only proteins Bim and Noxa and repressing Survivin [#5, #10], and enforces cell-cycle arrest via p27Kip1 [#9]. Beyond apoptosis, FOXO3a binds promoters of an antioxidant, metabolic, and autophagy program: it transactivates SIRT6 to suppress the Warburg effect [#33], drives Keap1 transcription to constrain Nrf2 [#16], activates BNIP3, PARKIN, and PERK to control mitophagy and the unfolded-protein response [#17, #27, #22], and represses SLC7A11 to limit ferroptosis [#29]. FOXO3 also coordinates the DNA-damage response by binding the ATM FAT domain and the ATM-Chk2-p53 complex to promote checkpoint activation, repair, and apoptosis [#8, #14]. Its own expression and localization are regulated by transcriptional activators (TEAD1, WTIP), m6A methylation (METTL14), nuclear-retention partners (SNAI2), and cytoplasmic-sequestering partners (REP1) [#13, #23, #31, #28, #26], and FOXO3 maintains the neural stem cell pool by enforcing quiescence and controlling oxygen metabolism [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established the core regulatory logic of FOXO3a: how survival kinases inactivate it through site-specific phosphorylation.\",\n      \"evidence\": \"In vitro kinase assays with site-directed mutagenesis of Thr-32/Ser-253/Ser-315 and reporter/survival assays in cells\",\n      \"pmids\": [\"11154281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which phosphatase reverses inactivation\", \"In vivo physiological relevance of distinct Akt vs SGK1 site usage not established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed the phosphorylation switch is engaged by physiological extracellular cues, linking FOXO3a to TGF-\\u03b2 and thrombopoietin signaling and identifying its transactivation domain as required for apoptosis.\",\n      \"evidence\": \"Triple Akt-site mutant, dominant-negative Akt and PI3K inhibitor in mammary epithelial cells; inducible constitutively active allele in TPO-dependent megakaryocytic cells\",\n      \"pmids\": [\"11694570\", \"11278373\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct target genes mediating arrest/apoptosis in these contexts not defined\", \"Megakaryocyte study is Medium confidence with limited methods\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Extended PI3K/Akt-mediated FOXO3a inactivation to neurotrophin-driven neuronal survival.\",\n      \"evidence\": \"Kinase inhibitors, constitutively active/dominant-negative Akt, in vitro kinase assay, TrkA vs p75 cell comparison in PC12 cells\",\n      \"pmids\": [\"11953455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream survival targets not identified\", \"Single cell-line model\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Revealed an Akt-independent route to FOXO3a inactivation during DNA damage, mediated by p53-induced SGK1.\",\n      \"evidence\": \"siRNA knockdown of SGK1, pharmacological inhibitors, localization and phosphorylation assays in p53-deficient cells\",\n      \"pmids\": [\"15383658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with FOXO3a's pro-apoptotic role in DNA damage not fully resolved\", \"Specific phosphosites engaged by p53/SGK1 not mapped\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the molecular effectors of FOXO3a-driven apoptosis, establishing it as a transcriptional trigger of the mitochondrial death pathway.\",\n      \"evidence\": \"4-OHT-regulated FKHRL1(A3)ER transgene, RNAi of Noxa/Bim, dnFADD, Bcl2 overexpression, cytochrome c and caspase assays; promoter binding to G6Pase IRSs\",\n      \"pmids\": [\"16888645\", \"16840535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect transactivation of all BH3-only genes not fully dissected\", \"Metabolic G6Pase study is Medium confidence\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified a non-transcriptional, protein-protein function for FOXO3a in the DNA-damage response via direct ATM activation.\",\n      \"evidence\": \"Domain-mapped Co-IP (FOXO3a C-terminus to ATM FAT domain), siRNA, foci immunofluorescence, checkpoint and repair assays\",\n      \"pmids\": [\"18344987\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the FOXO3a-FAT interface not determined\", \"How phosphorylation status of FOXO3a gates this interaction unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved how FOXO3a enforces quiescence and survival in proliferating cells, distinguishing late vs early cell-cycle control and defining its stem-cell role.\",\n      \"evidence\": \"Adenoviral overexpression with p27Kip1/p21 promoter-luciferase in muscle precursors; FoxO3 knockout mice with neurosphere, multipotency, and expression profiling\",\n      \"pmids\": [\"17894357\", \"19896443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct FOXO3 occupancy of p27Kip1 promoter not shown by ChIP in muscle study (Medium)\", \"NSC quiescence gene program members only partially defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed FOXO3a sensitizes cells to genotoxic therapy by repressing the anti-apoptotic node Survivin.\",\n      \"evidence\": \"Conditional FKHRL1(A3)ER allele, Survivin shRNA and transgenic rescue, mitochondrial readouts, DNA-damaging drug sensitivity\",\n      \"pmids\": [\"19211844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding of FOXO3a to BIRC5/Survivin regulatory regions not mapped\", \"Generality across tumor types untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established acetylation by p300 as a degradation/relocalization switch and identified TEAD1 as an upstream transcriptional activator of FoxO3a in muscle.\",\n      \"evidence\": \"Co-IP (FoxO3-p300), acetylation-mimic/resistant mutants (Lys-262) in denervation model; ChIP/EMSA/luciferase for TEAD1 at the FoxO3a M-CAT element\",\n      \"pmids\": [\"22094330\", \"21211055\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deacetylases acting on these lysines in muscle not identified in this study\", \"TEAD1 study is Medium confidence and muscle-restricted\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed FOXO3 inside the ATM-Chk2-p53 DNA-damage complex as an essential bidirectional partner and defined a FOXO3-ATM-CREB axis controlling caspase-8.\",\n      \"evidence\": \"Reciprocal Co-IP of the four-protein complex, siRNA epistasis, foci and apoptosis assays; conditional FOXO3 transgene with ATM/CREB inhibition and bisulfite methylation analysis\",\n      \"pmids\": [\"22893124\", \"22493319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the FOXO3-ATM-Chk2-p53 complex unresolved\", \"caspase-8 axis study is Medium confidence\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined FOXO3 as a transcriptional governor of redox balance through direct Keap1 activation and BNIP3-driven mitochondrial fate.\",\n      \"evidence\": \"ChIP, siRNA, luciferase and xenograft for the FoxO3-Keap1-Nrf2 axis; dominant-negative FOXO3a (AAV9) with BNIP3 manipulation and cardiac functional readouts\",\n      \"pmids\": [\"26857210\", \"27694219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between FOXO3-driven Keap1 (anti-Nrf2) and FOXO3's own antioxidant role not integrated\", \"Tissue-specific weighting of these outputs unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified prolyl hydroxylation as an oxygen-sensing degradation switch and established direct FOXO3 transactivation of metabolic/stress targets SIRT6 and PERK.\",\n      \"evidence\": \"Tubule-specific FoxO3 and HIF-1\\u03b1 conditional KO with prolyl hydroxylation assays and renal injury models; ChIP/luciferase/triple-KO MEF validation for SIRT6 and PERK targets; NMR-validated small molecules targeting the FOXO3 DNA-binding domain\",\n      \"pmids\": [\"30912765\", \"31004738\", \"32124950\", \"31312024\", \"31789593\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The PHD enzyme(s) and hydroxylated proline residues not all individually mapped\", \"Druggability shown in cells but not in vivo efficacy\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Confirmed direct FOXO3a transactivation of SIRT6 to suppress aerobic glycolysis across multiple cancer types.\",\n      \"evidence\": \"ChIP, luciferase, knockdown/overexpression epistasis with glycolysis assays and xenografts in melanoma and glioblastoma\",\n      \"pmids\": [\"32124950\", \"31004738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the FOXO3-SIRT6 axis operates in non-transformed metabolic tissues untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded FOXO3's transcriptional output to autophagy/mitophagy and revealed positive feedback loops and partner-controlled localization that amplify or constrain its activity.\",\n      \"evidence\": \"ChIP and feedback dissection for COPS3 and the SNAI2-FOXO3 feed-forward loop (with CRM1 export assays, conserved in Drosophila); Co-IP for SIRT1 (BNIP3/PINK1-Parkin) and REP1 (cytoplasmic sequestration); ChIP/luciferase for PARKIN with transgenic cardiac rescue\",\n      \"pmids\": [\"36451342\", \"35271390\", \"39266959\", \"28055019\", \"36539848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Several partner interactions rest on single-lab Co-IP (Medium)\", \"Hierarchy among competing nuclear-retention and export factors unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed FOXO3a controls non-canonical outputs\\u2014ferroptosis suppression via SLC7A11 repression, selective circRNA biogenesis (circSPON1), and its own m6A-dependent stability.\",\n      \"evidence\": \"ChIP and FOXO3a KO with AMPK/ferroptosis readouts (SLC7A11); ChIP plus selective circRNA expression and Co-IP (circSPON1-Smad3); m6A-seq/meRIP and mRNA stability assays (METTL14)\",\n      \"pmids\": [\"37267686\", \"37416778\", \"37935312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of FOXO3 selectivity for circRNA over mRNA at SPON1 not defined\", \"All three findings are single-lab Medium-confidence\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many competing post-translational modifications, partner interactions, and feedback loops are integrated to set FOXO3 nuclear residence and target-gene selection in a given cell state remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No quantitative model linking PTM combinations to specific target-gene programs\", \"Structural basis for FOXO3 partner discrimination unknown\", \"Mechanism of promoter selectivity (apoptosis vs autophagy vs metabolic targets) undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 5, 16, 19, 22, 28, 29, 33]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 13, 16, 19, 21, 29, 30, 33]},\n      {\"term_id\": \"GO:0003700\", \"supporting_discovery_ids\": [5, 9, 16, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 8, 12, 23, 26, 28]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 12, 26]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [14, 16, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 10, 14, 23]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [16, 19, 22, 27, 33]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8, 14]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [24, 25, 28, 31]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 9, 11]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [16, 18, 29]}\n    ],\n    \"complexes\": [\n      \"ATM-Chk2-p53 complex\"\n    ],\n    \"partners\": [\n      \"ATM\",\n      \"AKT1\",\n      \"SGK1\",\n      \"EP300\",\n      \"SIRT1\",\n      \"SNAI2\",\n      \"WTIP\",\n      \"REP1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}