{"gene":"PROM1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2000,"finding":"The human AC133 antigen (PROM1/CD133) is the human orthologue of mouse prominin: both proteins are selectively localized to plasma membrane protrusions (microvilli in epithelial cells, membrane protrusions in hematopoietic stem cells and transfected fibroblasts), and this subcellular localization does not depend on an epithelial phenotype.","method":"Immunofluorescence, immunoprecipitation, electron microscopy, ectopic expression in fibroblasts, flow cytometry of murine CD34+ bone marrow progenitors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (immunofluorescence, EM, immunoprecipitation, ectopic expression) in a single rigorous study establishing both localization and orthology","pmids":["10681530"],"is_preprint":false},{"year":1997,"finding":"AC133 (PROM1/CD133) encodes a novel 865-amino-acid, 5-transmembrane domain cell-surface glycoprotein with a predicted molecular weight of 97 kDa (detected at ~120 kDa, consistent with glycosylation), selectively expressed on CD34bright hematopoietic stem and progenitor cells; AC133-selected cells engraft in a fetal sheep transplantation model and support secondary engraftment, demonstrating long-term repopulating potential.","method":"cDNA cloning, flow cytometry, immunoprecipitation, in vivo fetal sheep transplantation model with secondary engraftment","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — cDNA cloning plus in vivo functional engraftment assay with secondary recipients, replicated in subsequent work","pmids":["9389720"],"is_preprint":false},{"year":2002,"finding":"A novel isoform, AC133-2 (lacking a 27-nucleotide exon via alternative splicing), is glycosylated and transported to the plasma membrane when expressed in 293 cells; AC133-2, not AC133-1, is the isoform expressed on hematopoietic stem cells from fetal liver, bone marrow, and peripheral blood. AC133-2 co-expresses with β1-integrin in the basal layer of neonatal epidermis, and AC133-2+/β1-integrin+ cells lose AC133-2 and gain involucrin upon differentiation.","method":"cDNA cloning, recombinant expression in 293 cells, flow cytometry, immunofluorescence, in vitro differentiation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — recombinant expression with biochemical characterization and functional differentiation assays in one study","pmids":["12042327"],"is_preprint":false},{"year":2003,"finding":"The human AC133/PROM1 gene contains at least 9 distinctive 5′-UTR exons producing at least 7 alternatively spliced 5′-UTR mRNA isoforms expressed in a tissue-dependent manner, driven by 5 alternative promoters; in vitro methylation of 2 of these promoters completely suppresses their transcriptional activity.","method":"5′-UTR isoform identification by cDNA analysis, luciferase reporter assay for promoter activity, in vitro methylation followed by reporter assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Strong — luciferase reporter with in vitro methylation directly demonstrates promoter regulation; multiple promoters validated","pmids":["14630820"],"is_preprint":false},{"year":2010,"finding":"The AC133 antibody epitope is not lost upon cancer stem cell differentiation due to loss of CD133 protein or mRNA, but rather due to a change in CD133 glycosylation; differentially glycosylated CD133 can still be detected on the membrane of differentiated tumor cells by cell-surface biotinylation. AC133 antibody can detect unglycosylated CD133 (bacterially expressed) and glycosylation mutants.","method":"Flow cytometry, promoter activity assay, RT-PCR for mRNA/splice variants, immunoblot for protein, cell-surface biotinylation, bacterial expression of unglycosylated CD133, glycosylation-site mutagenesis","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods including mutagenesis, cell-surface biotinylation, and bacterial expression in one study","pmids":["20068153"],"is_preprint":false},{"year":2011,"finding":"N-glycan biosynthesis is required for cell-surface AC133/CD133 recognition: inhibition of N-glycan precursor biosynthesis (tunicamycin) or generation of N-glycan-deficient CD133 mutants abolishes cell-surface AC133 detection. MGAT4C, a complex N-glycan processing enzyme, positively regulates cell-surface AC133 expression.","method":"Large-scale pooled RNAi screen, secondary orthogonal RNAi validation, tunicamycin treatment, N-glycan-site mutagenesis, flow cytometry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — genome-wide RNAi screen validated orthogonally, combined with pharmacological inhibition and mutagenesis in one study","pmids":["21937449"],"is_preprint":false},{"year":2008,"finding":"CD133 transcription in ovarian cancer is directly regulated by epigenetic modifications: promoter methylation inversely correlates with CD133 transcription, and treatment with DNA methyltransferase inhibitors plus histone deacetylase inhibitors synergistically increases cell-surface CD133 expression in sorted CD133− cells. CD133+ cells retain an unmethylated/less-methylated promoter state while CD133− progeny show increased promoter methylation.","method":"Flow cytometry, bisulfite sequencing of promoter CpG sites, treatment with 5-azacytidine and/or valproic acid, cell sorting","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — bisulfite sequencing with pharmacological rescue across sorted populations, multiple methods in one study","pmids":["18836486"],"is_preprint":false},{"year":2008,"finding":"CD133 promoter activity in glioblastoma is regulated by DNA hypomethylation: three proximal CpG-containing promoters (P1, P2, P3) were isolated; in vitro methylation of P1 significantly inactivates it; treatment with the demethylating agent 5-azacytidine and/or histone deacetylase inhibitor valproic acid restores CD133 mRNA expression; hypomethylation of CpG sites within P1, P2, P3 is observed in high-CD133 human glioblastoma tissues.","method":"Promoter cloning, luciferase reporter assay, in vitro methylation, 5-azacytidine and valproic acid treatment, bisulfite sequencing of human glioblastoma tissues","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — luciferase reporter with in vitro methylation, pharmacological rescue, and bisulfite sequencing in primary tumors","pmids":["18679414"],"is_preprint":false},{"year":2012,"finding":"Sp1 and Myc transcription factors directly regulate CD133 promoter activity in glioma stem cells: Sp1 binds its predicted sites (gel-shift assays, ChIP with supershift); overexpression of Sp1 or Myc increases CD133 minimal promoter-driven luciferase activity and CD133 protein levels; the Sp1 inhibitor mithramycin decreases promoter activity and CD133 levels. In CD133-negative cells, methyl-DNA-binding proteins MBD1, MBD2, and MeCP2 bind the methylated CpG island and repress transcription (ChIP).","method":"Luciferase reporter assay (5′ and 3′ deletion constructs), gel-shift assay with supershift and competitive inhibition, ChIP, overexpression and pharmacological inhibition","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding confirmed by gel-shift with supershift and ChIP, combined with functional promoter assays and rescue experiments","pmids":["22945648"],"is_preprint":false},{"year":2018,"finding":"Prominin-1 (PROM1/CD133) interacts with the type I TGF-β receptor ALK4 and synergistically induces phosphorylation of Smad2; Prom1 overexpression consistently down-regulates cholesterol metabolism-associated genes and reduces cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axon regrowth. Genetic deletion of Prom1 in mice inhibits axon regeneration in DRG cultures and the sciatic nerve.","method":"Co-immunoprecipitation (Prom1–ALK4 interaction), AAV-mediated gene delivery in vivo, Smad2 phosphorylation assay, gene expression profiling, genetic knockout (Prom1−/− mice), in vitro DRG axon regeneration assay, sciatic nerve injury model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — Co-IP of binding partner combined with genetic KO, in vivo rescue, and mechanistic pathway (Smad2 phosphorylation, cholesterol) in one study","pmids":["32554499"],"is_preprint":false},{"year":2018,"finding":"CD133 forms a complex with E-cadherin and β-catenin (detected by immunoprecipitation); knockdown of CD133 reduces β-catenin levels in basal conditions and after Wnt pathway activation, and reduces TCF/LEF promoter activation, indicating that CD133 acts as a permissive factor for β-catenin/Wnt signaling by preventing its cytoplasmic degradation. Loss of CD133 limits cell proliferation after cisplatin-induced injury and impairs nephrosphere generation while promoting senescence.","method":"Immunoprecipitation (CD133–E-cadherin–β-catenin complex), siRNA knockdown, TCF/LEF luciferase reporter assay, RNA sequencing, nephrosphere formation, senescence assay","journal":"Stem cells translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for complex, luciferase reporter for pathway activity, KD with functional readouts; single lab, multiple methods","pmids":["29431914"],"is_preprint":false},{"year":2019,"finding":"Cytoplasmic (recycling endosomal) CD133 interacts with HDAC6 and is transported to the pericentrosomal region via a dynein-based trafficking system after internalization; at the pericentrosomal region, CD133 captures GABARAP (an autophagy initiator) and inhibits GABARAP-mediated ULK1 activation, thereby suppressing autophagy initiation. Phosphorylation of CD133 tyrosine 828 by Src family kinases controls this trafficking route. Pericentrosomal CD133 also suppresses primary ciliogenesis and neurite outgrowth by inhibiting autophagy.","method":"Co-immunoprecipitation (CD133–HDAC6, CD133–GABARAP), immunofluorescence localization, dynein inhibition, Src kinase inhibition/activation, ULK1 activation assay, autophagy flux assay, primary cilium formation assay, neurite outgrowth assay","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple Co-IPs with organelle fractionation, kinase-pathway manipulation, and multiple functional readouts in one study","pmids":["30783186"],"is_preprint":false},{"year":2018,"finding":"Prominin-1 overexpression increases microvilli number and induces branched/knob-like microvilli morphologies in MDCK cells through interaction with PI3K and the Arp2/3 complex; mutation of tyrosine 828 impairs CD133 phosphorylation and abolishes these interactions and the altered microvillar phenotypes. Silencing of human prominin-1 in primary hematopoietic stem cells results in loss of uropod-associated microvilli.","method":"Overexpression and mutagenesis (Y828 phosphorylation site), Co-immunoprecipitation (prominin-1–PI3K, prominin-1–Arp2/3), high-resolution microscopy, siRNA silencing in primary hematopoietic stem cells","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — site-directed mutagenesis combined with Co-IP of binding partners and functional morphological readouts, plus silencing in primary cells","pmids":["30328220"],"is_preprint":false},{"year":2018,"finding":"Forced expression of CD133 in U87MG glioma cells increases expression of IL-1β and its downstream chemokines CCL3, CXCL3, and CXCL5, leading to increased neutrophil recruitment in vitro (trans-well) and in vivo (tumor xenograft), without apparent changes in cell growth or sphere formation.","method":"Ectopic overexpression of CD133, qRT-PCR and western blot for IL-1β and chemokines, in vitro trans-well neutrophil recruitment assay, in vivo tumor xenograft neutrophil recruitment assay","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression with in vitro and in vivo functional readouts; single lab, two orthogonal assays (in vitro trans-well + xenograft)","pmids":["28736425"],"is_preprint":false},{"year":2013,"finding":"Lentivirus-mediated shRNA silencing of CD133 in human GBM patient-derived neurospheres impairs self-renewal and tumorigenic capacity; CD133 appears in an interconvertible state, changing subcellular localization between cytoplasm and plasma membrane of neurosphere cells.","method":"Lentiviral shRNA knockdown, neurosphere self-renewal assay, in vivo tumorigenicity assay, immunofluorescence for subcellular localization","journal":"Stem cells (Dayton, Ohio)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lentiviral KD with functional self-renewal and in vivo tumorigenicity readouts, plus direct localization experiment; single lab","pmids":["23307586"],"is_preprint":false},{"year":2010,"finding":"CD133 suppresses neuroblastoma cell differentiation (neurite extension and differentiation marker expression) and promotes proliferation and tumorigenesis partly by repressing transcription of the neurotrophic receptor RET via p38MAPK and PI3K/Akt pathways; RET overexpression rescues CD133-related inhibition of neurite elongation. CD133 also maintains this differentiation suppression in tumor spheres.","method":"Overexpression and silencing of CD133 in neuroblastoma lines, differentiation assays (neurite extension, marker expression), colony formation, in vivo tumor formation, RET overexpression rescue, pharmacological inhibition of p38MAPK and PI3K/Akt","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with pathway inhibition and rescue experiments; single lab, multiple orthogonal functional readouts","pmids":["20818439"],"is_preprint":false},{"year":2018,"finding":"CD133 regulates microvesicle (MV) release from the plasma membrane in colon cancer cells: EGF-induced NF-κB activation upregulates CD133, and the amount and size of budding MVs depend on CD133 expression level. CD133 mediates this by regulating activities of small GTPases RhoA and Rac1. CD133-containing MVs deliver mutant KRAS to adjacent cells and activate KRAS downstream signaling, promoting migration, invasion, and chemoresistance to anti-EGFR drugs.","method":"CD133 overexpression/knockdown, MV quantification and sizing, RhoA/Rac1 activity assay, KRAS mutant transfer assay, NF-κB reporter, cell migration and invasion assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain/loss-of-function with GTPase activity assays and functional oncoprotein transfer readout; single lab, multiple orthogonal methods","pmids":["30521383"],"is_preprint":false},{"year":2020,"finding":"Aberrant PROM1/CD133 expression in MLL-AF4 infant/childhood ALL is essential for leukemic cell growth and is driven by direct MLL-AF4 binding; activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) that increases enhancer–promoter interactions between PROM1 and the nearby gene TAPT1. In PROM1-non-expressing cells the locus is repressed by PRC2 binding.","method":"ChIP-seq (MLL-AF4, H3K79me2/3, PRC2), Hi-C/promoter capture Hi-C (enhancer–promoter interaction), CRISPR/shRNA knockdown of PROM1 with cell growth readout, correlation analyses in leukemia datasets","journal":"Leukemia","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP-seq for direct binding, chromatin interaction mapping, and loss-of-function growth assay; multiple orthogonal methods in one study","pmids":["32242051"],"is_preprint":false},{"year":2015,"finding":"Nucleolin directly binds the tissue-dependent CD133 promoter P1 and activates AC133/CD133 expression in CD34+ hematopoietic stem/progenitor cells; nucleolin knockdown reduces AC133 surface expression, colony-forming unit frequencies, long-term culture-initiating cells, and β-catenin, Akt, and Bcl-2 levels; these effects partially depend on β-catenin activity.","method":"Nucleolin ChIP on CD133 P1 promoter, nucleolin knockdown/overexpression, flow cytometry of AC133, CFU assay, LTC-IC assay, western blot for β-catenin/Akt/Bcl-2","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for direct promoter binding with functional downstream readouts; single lab, two orthogonal approaches","pmids":["26183533"],"is_preprint":false},{"year":2000,"finding":"Circulating human CD34+ cells co-expressing VEGFR-2 and AC133 constitute a phenotypically distinct population of circulating endothelial precursors (CEPs): upon culture with VEGF/FGF-2, AC133+VEGFR-2+ cells differentiate into AC133−VEGFR-2+Ac-LDL+ endothelial colonies (3% plating efficiency); mature endothelial cells do not express AC133, indicating loss of AC133 marks terminal endothelial differentiation. In vivo, AC133+VEGFR-2+ cells were found in neo-intima on left ventricular assist devices.","method":"Flow cytometry, in vitro differentiation assay (VEGF/FGF-2 culture, Ac-LDL uptake), in vivo examination of left ventricular assist device neo-intima","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional in vitro differentiation assay with in vivo corroboration; single lab, two orthogonal readouts","pmids":["10648408"],"is_preprint":false},{"year":2014,"finding":"Genetic deletion of Prom1 in mice increases susceptibility to intestinal tumor formation; in APC-mutant mice, Prom1-expressing cells are increased in intestinal crypt stem cell compartments and early adenomas, indicating a role for Prom1 in regulating intestinal homeostasis and tumor suppression in this context.","method":"Prom1 knockout mice, APC-mutant mouse model, intestinal tumor formation assay, immunohistochemistry","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with in vivo tumor formation readout; single lab, clean phenotypic assay","pmids":["25452936"],"is_preprint":false},{"year":2007,"finding":"A homozygous nonsense mutation in PROM1 (c.1726C>T, p.Gln576X) causes severe autosomal recessive retinitis pigmentosa with macular degeneration, mapping to chromosome 4p14-p16; rod and cone ERG responses are extinguished, establishing loss-of-function of PROM1 as causative for severe photoreceptor degeneration.","method":"Genome-wide linkage scan, direct sequencing of PROM1 exons, segregation analysis in family, ERG recordings","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — linkage plus segregating truncating mutation with functional ERG readout; establishes loss-of-function consequence but no molecular mechanism","pmids":["17605048"],"is_preprint":false},{"year":2019,"finding":"Recessive PROM1 truncating and splice-site variants result in early-onset severe panretinal cone-rod dystrophy with macular involvement, while a dominant c.1117C>T missense variant causes a milder, cone-predominant macular phenotype; the similar severity of homozygous missense vs. truncating recessive variants suggests a null or near-null outcome for all recessive alleles.","method":"Next-generation sequencing, ophthalmic examination, retinal imaging, ERG, segregation analysis across 19 patients","journal":"JAMA network open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic genotype–phenotype correlation across 19 patients with NGS and functional ERG; multiple allele types compared","pmids":["31199449"],"is_preprint":false},{"year":2018,"finding":"A truncated, labile, and mislocalized PROM1 protein results from the c.C1902G (p.Y634X) nonsense mutation associated with macular and rod-cone dystrophy, confirmed by expression in cultured cells and confocal microscopy; a second c.C1682+3A>G intronic mutation disrupts mRNA splicing (confirmed by bridge-PCR).","method":"Whole exome sequencing, transient transfection and expression in cultured cells, confocal microscopy for protein localization, bridge-PCR for splice disruption","journal":"Graefe's archive for clinical and experimental ophthalmology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based expression of mutant protein with localization readout and molecular splicing assay; two orthogonal methods, single lab","pmids":["30588538"],"is_preprint":false}],"current_model":"PROM1/CD133 is a pentaspan transmembrane glycoprotein that selectively localizes to plasma membrane protrusions (microvilli, uropods) via interactions with PI3K and the Arp2/3 complex; its N-glycosylation state determines recognition by the AC133 antibody epitope and governs cell-surface versus intracellular distribution. Transcription is driven by tissue-specific alternative promoters regulated by DNA methylation (enforced by MBD1/2/MeCP2) and by transcription factors Sp1, Myc, and nucleolin. Intracellularly, CD133 undergoes dynein/HDAC6-dependent trafficking to the pericentrosomal recycling endosome where it sequesters GABARAP to suppress ULK1-mediated autophagy initiation, thereby maintaining an undifferentiated state and suppressing ciliogenesis. At the plasma membrane, CD133 forms a complex with E-cadherin and β-catenin and promotes Wnt/β-catenin signaling by protecting β-catenin from degradation; it also modulates microvesicle release via RhoA/Rac1 GTPases. In neurons, prominin-1 binds ALK4 to synergistically phosphorylate Smad2 and down-regulate cholesterol biosynthesis, thereby promoting axon regeneration. In the retina, loss-of-function of PROM1 causes photoreceptor degeneration (retinitis pigmentosa and cone-rod dystrophy)."},"narrative":{"mechanistic_narrative":"PROM1/CD133 is a pentaspan transmembrane glycoprotein marking stem and progenitor populations across hematopoietic, epithelial, neural, and endothelial lineages, where it selectively concentrates in plasma-membrane protrusions and is lost upon differentiation [PMID:9389720, PMID:10681530, PMID:12042327]. At the cell surface it shapes membrane architecture: phosphorylation of tyrosine 828 by Src-family kinases drives interactions with PI3K and the Arp2/3 complex to increase and remodel microvilli, and silencing prominin-1 abolishes uropod-associated microvilli in hematopoietic stem cells [PMID:30328220]. Its detection by the AC133 antibody is governed by N-glycosylation rather than protein abundance, with complex N-glycan processing (MGAT4C) and glycosylation state determining cell-surface epitope recognition and intracellular versus surface distribution [PMID:20068153, PMID:21937449]. The same Y828 trafficking switch routes internalized CD133 by dynein/HDAC6-dependent transport to the pericentrosomal recycling endosome, where it sequesters GABARAP to block ULK1-mediated autophagy initiation and thereby suppresses ciliogenesis and neurite outgrowth, maintaining an undifferentiated state [PMID:30783186]. CD133 reinforces stemness through signaling: it forms a complex with E-cadherin and β-catenin that protects β-catenin from degradation and sustains TCF/LEF transcription [PMID:29431914], and in neurons binds the TGF-β type I receptor ALK4 to potentiate Smad2 phosphorylation and lower cholesterol biosynthesis, promoting axon regeneration [PMID:32554499]. Transcription of PROM1 is driven by multiple tissue-specific alternative promoters under epigenetic control, repressed by promoter CpG methylation and methyl-CpG-binding proteins (MBD1/2, MeCP2) and activated by Sp1, Myc, nucleolin, and in MLL-rearranged leukemia by an MLL-AF4-bound intragenic enhancer [PMID:14630820, PMID:18836486, PMID:22945648, PMID:26183533, PMID:32242051]. Loss-of-function PROM1 mutations cause autosomal recessive retinitis pigmentosa and cone-rod dystrophy with macular degeneration [PMID:17605048, PMID:31199449].","teleology":[{"year":1997,"claim":"Establishing that AC133 is a novel 5-transmembrane stem-cell surface glycoprotein gave the field a defined molecular marker of long-term repopulating hematopoietic stem cells.","evidence":"cDNA cloning and flow cytometry plus in vivo fetal sheep transplantation with secondary engraftment","pmids":["9389720"],"confidence":"High","gaps":["No molecular function assigned beyond marker status","Mechanism linking expression to repopulating potential unknown"]},{"year":2000,"claim":"Demonstrating that human AC133 is the orthologue of mouse prominin and selectively localizes to plasma-membrane protrusions independent of epithelial phenotype defined its conserved subcellular localization principle.","evidence":"Immunofluorescence, EM, immunoprecipitation, and ectopic expression in fibroblasts; parallel identification of CD34+VEGFR-2+ endothelial precursors losing AC133 on differentiation","pmids":["10681530","10648408"],"confidence":"High","gaps":["Molecular determinants of protrusion targeting not yet defined","Functional consequence of protrusion localization unknown"]},{"year":2003,"claim":"Mapping multiple tissue-specific alternative promoters and showing methylation-silenced isoforms explained how a single locus achieves lineage-restricted expression.","evidence":"5'-UTR isoform cloning, luciferase reporter assays, and in vitro promoter methylation; alternative splicing producing the surface-targeted AC133-2 isoform","pmids":["14630820","12042327"],"confidence":"High","gaps":["Which transcription factors drive each promoter not yet identified","In vivo relevance of individual isoforms unresolved"]},{"year":2008,"claim":"Linking promoter CpG methylation state to CD133 expression in ovarian cancer and glioblastoma established epigenetic control as the switch between marker-positive and marker-negative tumor populations.","evidence":"Bisulfite sequencing, in vitro promoter methylation, and pharmacological demethylation/HDAC inhibition across sorted populations and primary tumors","pmids":["18836486","18679414"],"confidence":"High","gaps":["Trans-acting factors reading methylation state not defined here","Whether methylation changes are cause or consequence of differentiation unclear"]},{"year":2010,"claim":"Showing that AC133 epitope loss reflects altered glycosylation rather than protein loss resolved a major confound in cancer-stem-cell marker interpretation.","evidence":"Cell-surface biotinylation, bacterial expression of unglycosylated CD133, and glycosylation-site mutagenesis with flow cytometry; functional differentiation suppression demonstrated in neuroblastoma","pmids":["20068153","20818439"],"confidence":"High","gaps":["Functional role of differential glycosylation beyond epitope masking unknown","Glycosyltransferases responsible not identified in this study"]},{"year":2012,"claim":"Identifying Sp1, Myc, and methyl-CpG-binding repressors (MBD1/2, MeCP2), and later nucleolin, as direct promoter regulators connected the epigenetic state to a defined transcription-factor network.","evidence":"Gel-shift with supershift, ChIP, reporter assays with TF overexpression and pharmacological inhibition (2012); nucleolin ChIP on promoter P1 with functional HSPC readouts (2015)","pmids":["22945648","26183533"],"confidence":"High","gaps":["Combinatorial logic of these factors at distinct promoters unresolved","Direct nucleolin mechanism on chromatin not detailed"]},{"year":2011,"claim":"A genome-wide RNAi screen establishing that N-glycan biosynthesis and MGAT4C are required for surface AC133 recognition defined the post-translational basis of epitope display.","evidence":"Pooled RNAi screen with orthogonal validation, tunicamycin treatment, and N-glycan-site mutagenesis with flow cytometry","pmids":["21937449"],"confidence":"High","gaps":["How glycosylation alters trafficking versus folding not separated","Physiological signal controlling glycan state unknown"]},{"year":2018,"claim":"Defining Y828-phosphorylation-dependent interactions with PI3K/Arp2/3 (membrane), ALK4/Smad2 (neurons), and E-cadherin/β-catenin (Wnt) converted CD133 from a passive marker into an active signaling and morphogenetic effector.","evidence":"Site-directed Y828 mutagenesis with Co-IP and microvillar morphology (Traffic); Co-IP plus Prom1 KO and in vivo axon regeneration (PNAS); Co-IP plus knockdown with TCF/LEF reporter (Stem Cells Transl Med)","pmids":["30328220","32554499","29431914"],"confidence":"High","gaps":["Reciprocal validation of E-cadherin/β-catenin complex limited to single lab","How one Y828 phospho-switch coordinates divergent partner choices unresolved"]},{"year":2019,"claim":"Showing that dynein/HDAC6-dependent pericentrosomal trafficking lets CD133 sequester GABARAP to block ULK1 and suppress autophagy, ciliogenesis, and neurite outgrowth provided a mechanistic route by which CD133 enforces an undifferentiated state.","evidence":"Co-IP (CD133-HDAC6, CD133-GABARAP), dynein/Src inhibition, ULK1 and autophagy-flux assays, cilium and neurite readouts","pmids":["30783186"],"confidence":"High","gaps":["Quantitative contribution of autophagy suppression to stemness in vivo not measured","Interplay between surface signaling and endosomal pool unresolved"]},{"year":2020,"claim":"Showing CD133 controls microvesicle release via RhoA/Rac1 and is itself driven by an MLL-AF4-bound intragenic enhancer in leukemia extended its roles to intercellular oncoprotein transfer and a defined oncogenic transcriptional circuit.","evidence":"Gain/loss-of-function with GTPase activity and KRAS-transfer assays (FASEB, 2018); ChIP-seq, Hi-C, and CRISPR/shRNA growth assays in MLL-AF4 ALL (Leukemia, 2020)","pmids":["30521383","32242051"],"confidence":"Medium","gaps":["Microvesicle GTPase mechanism from single lab","Whether enhancer dependence generalizes beyond MLL-rearranged leukemia unknown"]},{"year":2019,"claim":"Identifying recessive null and dominant missense PROM1 mutations as causes of photoreceptor degeneration linked loss of CD133 function directly to human retinal disease.","evidence":"Linkage and segregation analysis, NGS, ERG, and mutant-protein expression with confocal localization across families and a 19-patient cohort","pmids":["17605048","31199449","30588538"],"confidence":"Medium","gaps":["Molecular role of PROM1 in photoreceptor outer-segment biology not mechanistically defined in corpus","How truncated/mislocalized protein causes degeneration unresolved"]},{"year":null,"claim":"How the single Y828 phospho-switch and glycosylation state are coordinated to partition CD133 between surface morphogenetic/signaling functions and the endosomal autophagy-suppressing pool, and how this governs differentiation in normal versus malignant stem cells, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating surface, signaling, and endosomal roles","Structural basis of partner selection unknown","Direct in vivo link from autophagy/Wnt regulation to retinal degeneration not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[9,11,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,16]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,12]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,2,12]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[11,14]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,10]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[11]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[3,6,7,8,17,18]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,5]}],"complexes":[],"partners":["CDH1","CTNNB1","ACVR1B","GABARAP","HDAC6","PIK3","ARPC2","NCL"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43490","full_name":"Prominin-1","aliases":["Antigen AC133","Prominin-like protein 1"],"length_aa":865,"mass_kda":97.2,"function":"May play a role in cell differentiation, proliferation and apoptosis (PubMed:24556617). Binds cholesterol in cholesterol-containing plasma membrane microdomains and may play a role in the organization of the apical plasma membrane in epithelial cells. During early retinal development acts as a key regulator of disk morphogenesis. Involved in regulation of MAPK and Akt signaling pathways. 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/20428792","citation_count":26,"is_preprint":false},{"pmid":"20859302","id":"PMC_20859302","title":"Extended extraocular phenotype of PROM1 mutation in kindreds with known autosomal dominant macular dystrophy.","date":"2010","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/20859302","citation_count":26,"is_preprint":false},{"pmid":"26183533","id":"PMC_26183533","title":"Control of AC133/CD133 and impact on human hematopoietic progenitor cells through nucleolin.","date":"2015","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/26183533","citation_count":24,"is_preprint":false},{"pmid":"28819416","id":"PMC_28819416","title":"Expression of CD133 in endometrial cancer cells and its implications.","date":"2017","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/28819416","citation_count":24,"is_preprint":false},{"pmid":"25014242","id":"PMC_25014242","title":"The CD133+ cell as advanced medicinal product for myocardial and limb ischemia.","date":"2014","source":"Stem cells and development","url":"https://pubmed.ncbi.nlm.nih.gov/25014242","citation_count":23,"is_preprint":false},{"pmid":"23545719","id":"PMC_23545719","title":"Expression and localisation of osteopontin and prominin-1 (CD133) in patients with endometriosis.","date":"2013","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23545719","citation_count":22,"is_preprint":false},{"pmid":"23161082","id":"PMC_23161082","title":"Prominin-1 (CD133) Expression in the Prostate and Prostate Cancer: A Marker for Quiescent Stem Cells.","date":"2013","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/23161082","citation_count":22,"is_preprint":false},{"pmid":"19327159","id":"PMC_19327159","title":"AC133+ progenitor cells as gene delivery vehicle and cellular probe in subcutaneous tumor models: a preliminary study.","date":"2009","source":"BMC biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/19327159","citation_count":22,"is_preprint":false},{"pmid":"30783186","id":"PMC_30783186","title":"Recycling endosomal CD133 functions as an inhibitor of autophagy at the pericentrosomal region.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30783186","citation_count":22,"is_preprint":false},{"pmid":"26137114","id":"PMC_26137114","title":"Detection of CD133 expression in U87 glioblastoma cells using a novel anti-CD133 monoclonal antibody.","date":"2015","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/26137114","citation_count":22,"is_preprint":false},{"pmid":"35785618","id":"PMC_35785618","title":"Single-cell sequencing reveals CD133+CD44--originating evolution and novel stemness related variants in human colorectal cancer.","date":"2022","source":"EBioMedicine","url":"https://pubmed.ncbi.nlm.nih.gov/35785618","citation_count":21,"is_preprint":false},{"pmid":"30588538","id":"PMC_30588538","title":"Identification of novel PROM1 mutations responsible for autosomal recessive maculopathy with rod-cone dystrophy.","date":"2018","source":"Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie","url":"https://pubmed.ncbi.nlm.nih.gov/30588538","citation_count":21,"is_preprint":false},{"pmid":"32173531","id":"PMC_32173531","title":"Inhibition of NOTCH signaling pathway chemosensitizes HCC CD133+ cells to vincristine and 5-fluorouracil through upregulation of BBC3.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/32173531","citation_count":21,"is_preprint":false},{"pmid":"24271022","id":"PMC_24271022","title":"Isolation, identification and expression of specific human CD133 antibodies.","date":"2013","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/24271022","citation_count":20,"is_preprint":false},{"pmid":"27103380","id":"PMC_27103380","title":"CD133+ cells in pulmonary arterial hypertension.","date":"2016","source":"The European respiratory journal","url":"https://pubmed.ncbi.nlm.nih.gov/27103380","citation_count":19,"is_preprint":false},{"pmid":"25973297","id":"PMC_25973297","title":"Prominin-1 (CD133, AC133) and dipeptidyl-peptidase IV (CD26) are indicators of infinitive growth in colon cancer cells.","date":"2015","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/25973297","citation_count":19,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49329,"output_tokens":6927,"usd":0.125946,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16057,"output_tokens":4367,"usd":0.09473,"stage2_stop_reason":"end_turn"},"total_usd":0.220676,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"The human AC133 antigen (PROM1/CD133) is the human orthologue of mouse prominin: both proteins are selectively localized to plasma membrane protrusions (microvilli in epithelial cells, membrane protrusions in hematopoietic stem cells and transfected fibroblasts), and this subcellular localization does not depend on an epithelial phenotype.\",\n      \"method\": \"Immunofluorescence, immunoprecipitation, electron microscopy, ectopic expression in fibroblasts, flow cytometry of murine CD34+ bone marrow progenitors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (immunofluorescence, EM, immunoprecipitation, ectopic expression) in a single rigorous study establishing both localization and orthology\",\n      \"pmids\": [\"10681530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"AC133 (PROM1/CD133) encodes a novel 865-amino-acid, 5-transmembrane domain cell-surface glycoprotein with a predicted molecular weight of 97 kDa (detected at ~120 kDa, consistent with glycosylation), selectively expressed on CD34bright hematopoietic stem and progenitor cells; AC133-selected cells engraft in a fetal sheep transplantation model and support secondary engraftment, demonstrating long-term repopulating potential.\",\n      \"method\": \"cDNA cloning, flow cytometry, immunoprecipitation, in vivo fetal sheep transplantation model with secondary engraftment\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — cDNA cloning plus in vivo functional engraftment assay with secondary recipients, replicated in subsequent work\",\n      \"pmids\": [\"9389720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"A novel isoform, AC133-2 (lacking a 27-nucleotide exon via alternative splicing), is glycosylated and transported to the plasma membrane when expressed in 293 cells; AC133-2, not AC133-1, is the isoform expressed on hematopoietic stem cells from fetal liver, bone marrow, and peripheral blood. AC133-2 co-expresses with β1-integrin in the basal layer of neonatal epidermis, and AC133-2+/β1-integrin+ cells lose AC133-2 and gain involucrin upon differentiation.\",\n      \"method\": \"cDNA cloning, recombinant expression in 293 cells, flow cytometry, immunofluorescence, in vitro differentiation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — recombinant expression with biochemical characterization and functional differentiation assays in one study\",\n      \"pmids\": [\"12042327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The human AC133/PROM1 gene contains at least 9 distinctive 5′-UTR exons producing at least 7 alternatively spliced 5′-UTR mRNA isoforms expressed in a tissue-dependent manner, driven by 5 alternative promoters; in vitro methylation of 2 of these promoters completely suppresses their transcriptional activity.\",\n      \"method\": \"5′-UTR isoform identification by cDNA analysis, luciferase reporter assay for promoter activity, in vitro methylation followed by reporter assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — luciferase reporter with in vitro methylation directly demonstrates promoter regulation; multiple promoters validated\",\n      \"pmids\": [\"14630820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The AC133 antibody epitope is not lost upon cancer stem cell differentiation due to loss of CD133 protein or mRNA, but rather due to a change in CD133 glycosylation; differentially glycosylated CD133 can still be detected on the membrane of differentiated tumor cells by cell-surface biotinylation. AC133 antibody can detect unglycosylated CD133 (bacterially expressed) and glycosylation mutants.\",\n      \"method\": \"Flow cytometry, promoter activity assay, RT-PCR for mRNA/splice variants, immunoblot for protein, cell-surface biotinylation, bacterial expression of unglycosylated CD133, glycosylation-site mutagenesis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods including mutagenesis, cell-surface biotinylation, and bacterial expression in one study\",\n      \"pmids\": [\"20068153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"N-glycan biosynthesis is required for cell-surface AC133/CD133 recognition: inhibition of N-glycan precursor biosynthesis (tunicamycin) or generation of N-glycan-deficient CD133 mutants abolishes cell-surface AC133 detection. MGAT4C, a complex N-glycan processing enzyme, positively regulates cell-surface AC133 expression.\",\n      \"method\": \"Large-scale pooled RNAi screen, secondary orthogonal RNAi validation, tunicamycin treatment, N-glycan-site mutagenesis, flow cytometry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genome-wide RNAi screen validated orthogonally, combined with pharmacological inhibition and mutagenesis in one study\",\n      \"pmids\": [\"21937449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CD133 transcription in ovarian cancer is directly regulated by epigenetic modifications: promoter methylation inversely correlates with CD133 transcription, and treatment with DNA methyltransferase inhibitors plus histone deacetylase inhibitors synergistically increases cell-surface CD133 expression in sorted CD133− cells. CD133+ cells retain an unmethylated/less-methylated promoter state while CD133− progeny show increased promoter methylation.\",\n      \"method\": \"Flow cytometry, bisulfite sequencing of promoter CpG sites, treatment with 5-azacytidine and/or valproic acid, cell sorting\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — bisulfite sequencing with pharmacological rescue across sorted populations, multiple methods in one study\",\n      \"pmids\": [\"18836486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CD133 promoter activity in glioblastoma is regulated by DNA hypomethylation: three proximal CpG-containing promoters (P1, P2, P3) were isolated; in vitro methylation of P1 significantly inactivates it; treatment with the demethylating agent 5-azacytidine and/or histone deacetylase inhibitor valproic acid restores CD133 mRNA expression; hypomethylation of CpG sites within P1, P2, P3 is observed in high-CD133 human glioblastoma tissues.\",\n      \"method\": \"Promoter cloning, luciferase reporter assay, in vitro methylation, 5-azacytidine and valproic acid treatment, bisulfite sequencing of human glioblastoma tissues\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — luciferase reporter with in vitro methylation, pharmacological rescue, and bisulfite sequencing in primary tumors\",\n      \"pmids\": [\"18679414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Sp1 and Myc transcription factors directly regulate CD133 promoter activity in glioma stem cells: Sp1 binds its predicted sites (gel-shift assays, ChIP with supershift); overexpression of Sp1 or Myc increases CD133 minimal promoter-driven luciferase activity and CD133 protein levels; the Sp1 inhibitor mithramycin decreases promoter activity and CD133 levels. In CD133-negative cells, methyl-DNA-binding proteins MBD1, MBD2, and MeCP2 bind the methylated CpG island and repress transcription (ChIP).\",\n      \"method\": \"Luciferase reporter assay (5′ and 3′ deletion constructs), gel-shift assay with supershift and competitive inhibition, ChIP, overexpression and pharmacological inhibition\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding confirmed by gel-shift with supershift and ChIP, combined with functional promoter assays and rescue experiments\",\n      \"pmids\": [\"22945648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Prominin-1 (PROM1/CD133) interacts with the type I TGF-β receptor ALK4 and synergistically induces phosphorylation of Smad2; Prom1 overexpression consistently down-regulates cholesterol metabolism-associated genes and reduces cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axon regrowth. Genetic deletion of Prom1 in mice inhibits axon regeneration in DRG cultures and the sciatic nerve.\",\n      \"method\": \"Co-immunoprecipitation (Prom1–ALK4 interaction), AAV-mediated gene delivery in vivo, Smad2 phosphorylation assay, gene expression profiling, genetic knockout (Prom1−/− mice), in vitro DRG axon regeneration assay, sciatic nerve injury model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — Co-IP of binding partner combined with genetic KO, in vivo rescue, and mechanistic pathway (Smad2 phosphorylation, cholesterol) in one study\",\n      \"pmids\": [\"32554499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD133 forms a complex with E-cadherin and β-catenin (detected by immunoprecipitation); knockdown of CD133 reduces β-catenin levels in basal conditions and after Wnt pathway activation, and reduces TCF/LEF promoter activation, indicating that CD133 acts as a permissive factor for β-catenin/Wnt signaling by preventing its cytoplasmic degradation. Loss of CD133 limits cell proliferation after cisplatin-induced injury and impairs nephrosphere generation while promoting senescence.\",\n      \"method\": \"Immunoprecipitation (CD133–E-cadherin–β-catenin complex), siRNA knockdown, TCF/LEF luciferase reporter assay, RNA sequencing, nephrosphere formation, senescence assay\",\n      \"journal\": \"Stem cells translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for complex, luciferase reporter for pathway activity, KD with functional readouts; single lab, multiple methods\",\n      \"pmids\": [\"29431914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cytoplasmic (recycling endosomal) CD133 interacts with HDAC6 and is transported to the pericentrosomal region via a dynein-based trafficking system after internalization; at the pericentrosomal region, CD133 captures GABARAP (an autophagy initiator) and inhibits GABARAP-mediated ULK1 activation, thereby suppressing autophagy initiation. Phosphorylation of CD133 tyrosine 828 by Src family kinases controls this trafficking route. Pericentrosomal CD133 also suppresses primary ciliogenesis and neurite outgrowth by inhibiting autophagy.\",\n      \"method\": \"Co-immunoprecipitation (CD133–HDAC6, CD133–GABARAP), immunofluorescence localization, dynein inhibition, Src kinase inhibition/activation, ULK1 activation assay, autophagy flux assay, primary cilium formation assay, neurite outgrowth assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple Co-IPs with organelle fractionation, kinase-pathway manipulation, and multiple functional readouts in one study\",\n      \"pmids\": [\"30783186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Prominin-1 overexpression increases microvilli number and induces branched/knob-like microvilli morphologies in MDCK cells through interaction with PI3K and the Arp2/3 complex; mutation of tyrosine 828 impairs CD133 phosphorylation and abolishes these interactions and the altered microvillar phenotypes. Silencing of human prominin-1 in primary hematopoietic stem cells results in loss of uropod-associated microvilli.\",\n      \"method\": \"Overexpression and mutagenesis (Y828 phosphorylation site), Co-immunoprecipitation (prominin-1–PI3K, prominin-1–Arp2/3), high-resolution microscopy, siRNA silencing in primary hematopoietic stem cells\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — site-directed mutagenesis combined with Co-IP of binding partners and functional morphological readouts, plus silencing in primary cells\",\n      \"pmids\": [\"30328220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Forced expression of CD133 in U87MG glioma cells increases expression of IL-1β and its downstream chemokines CCL3, CXCL3, and CXCL5, leading to increased neutrophil recruitment in vitro (trans-well) and in vivo (tumor xenograft), without apparent changes in cell growth or sphere formation.\",\n      \"method\": \"Ectopic overexpression of CD133, qRT-PCR and western blot for IL-1β and chemokines, in vitro trans-well neutrophil recruitment assay, in vivo tumor xenograft neutrophil recruitment assay\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression with in vitro and in vivo functional readouts; single lab, two orthogonal assays (in vitro trans-well + xenograft)\",\n      \"pmids\": [\"28736425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Lentivirus-mediated shRNA silencing of CD133 in human GBM patient-derived neurospheres impairs self-renewal and tumorigenic capacity; CD133 appears in an interconvertible state, changing subcellular localization between cytoplasm and plasma membrane of neurosphere cells.\",\n      \"method\": \"Lentiviral shRNA knockdown, neurosphere self-renewal assay, in vivo tumorigenicity assay, immunofluorescence for subcellular localization\",\n      \"journal\": \"Stem cells (Dayton, Ohio)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lentiviral KD with functional self-renewal and in vivo tumorigenicity readouts, plus direct localization experiment; single lab\",\n      \"pmids\": [\"23307586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CD133 suppresses neuroblastoma cell differentiation (neurite extension and differentiation marker expression) and promotes proliferation and tumorigenesis partly by repressing transcription of the neurotrophic receptor RET via p38MAPK and PI3K/Akt pathways; RET overexpression rescues CD133-related inhibition of neurite elongation. CD133 also maintains this differentiation suppression in tumor spheres.\",\n      \"method\": \"Overexpression and silencing of CD133 in neuroblastoma lines, differentiation assays (neurite extension, marker expression), colony formation, in vivo tumor formation, RET overexpression rescue, pharmacological inhibition of p38MAPK and PI3K/Akt\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with pathway inhibition and rescue experiments; single lab, multiple orthogonal functional readouts\",\n      \"pmids\": [\"20818439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CD133 regulates microvesicle (MV) release from the plasma membrane in colon cancer cells: EGF-induced NF-κB activation upregulates CD133, and the amount and size of budding MVs depend on CD133 expression level. CD133 mediates this by regulating activities of small GTPases RhoA and Rac1. CD133-containing MVs deliver mutant KRAS to adjacent cells and activate KRAS downstream signaling, promoting migration, invasion, and chemoresistance to anti-EGFR drugs.\",\n      \"method\": \"CD133 overexpression/knockdown, MV quantification and sizing, RhoA/Rac1 activity assay, KRAS mutant transfer assay, NF-κB reporter, cell migration and invasion assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain/loss-of-function with GTPase activity assays and functional oncoprotein transfer readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"30521383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Aberrant PROM1/CD133 expression in MLL-AF4 infant/childhood ALL is essential for leukemic cell growth and is driven by direct MLL-AF4 binding; activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) that increases enhancer–promoter interactions between PROM1 and the nearby gene TAPT1. In PROM1-non-expressing cells the locus is repressed by PRC2 binding.\",\n      \"method\": \"ChIP-seq (MLL-AF4, H3K79me2/3, PRC2), Hi-C/promoter capture Hi-C (enhancer–promoter interaction), CRISPR/shRNA knockdown of PROM1 with cell growth readout, correlation analyses in leukemia datasets\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP-seq for direct binding, chromatin interaction mapping, and loss-of-function growth assay; multiple orthogonal methods in one study\",\n      \"pmids\": [\"32242051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Nucleolin directly binds the tissue-dependent CD133 promoter P1 and activates AC133/CD133 expression in CD34+ hematopoietic stem/progenitor cells; nucleolin knockdown reduces AC133 surface expression, colony-forming unit frequencies, long-term culture-initiating cells, and β-catenin, Akt, and Bcl-2 levels; these effects partially depend on β-catenin activity.\",\n      \"method\": \"Nucleolin ChIP on CD133 P1 promoter, nucleolin knockdown/overexpression, flow cytometry of AC133, CFU assay, LTC-IC assay, western blot for β-catenin/Akt/Bcl-2\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for direct promoter binding with functional downstream readouts; single lab, two orthogonal approaches\",\n      \"pmids\": [\"26183533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Circulating human CD34+ cells co-expressing VEGFR-2 and AC133 constitute a phenotypically distinct population of circulating endothelial precursors (CEPs): upon culture with VEGF/FGF-2, AC133+VEGFR-2+ cells differentiate into AC133−VEGFR-2+Ac-LDL+ endothelial colonies (3% plating efficiency); mature endothelial cells do not express AC133, indicating loss of AC133 marks terminal endothelial differentiation. In vivo, AC133+VEGFR-2+ cells were found in neo-intima on left ventricular assist devices.\",\n      \"method\": \"Flow cytometry, in vitro differentiation assay (VEGF/FGF-2 culture, Ac-LDL uptake), in vivo examination of left ventricular assist device neo-intima\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional in vitro differentiation assay with in vivo corroboration; single lab, two orthogonal readouts\",\n      \"pmids\": [\"10648408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Genetic deletion of Prom1 in mice increases susceptibility to intestinal tumor formation; in APC-mutant mice, Prom1-expressing cells are increased in intestinal crypt stem cell compartments and early adenomas, indicating a role for Prom1 in regulating intestinal homeostasis and tumor suppression in this context.\",\n      \"method\": \"Prom1 knockout mice, APC-mutant mouse model, intestinal tumor formation assay, immunohistochemistry\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with in vivo tumor formation readout; single lab, clean phenotypic assay\",\n      \"pmids\": [\"25452936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A homozygous nonsense mutation in PROM1 (c.1726C>T, p.Gln576X) causes severe autosomal recessive retinitis pigmentosa with macular degeneration, mapping to chromosome 4p14-p16; rod and cone ERG responses are extinguished, establishing loss-of-function of PROM1 as causative for severe photoreceptor degeneration.\",\n      \"method\": \"Genome-wide linkage scan, direct sequencing of PROM1 exons, segregation analysis in family, ERG recordings\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — linkage plus segregating truncating mutation with functional ERG readout; establishes loss-of-function consequence but no molecular mechanism\",\n      \"pmids\": [\"17605048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Recessive PROM1 truncating and splice-site variants result in early-onset severe panretinal cone-rod dystrophy with macular involvement, while a dominant c.1117C>T missense variant causes a milder, cone-predominant macular phenotype; the similar severity of homozygous missense vs. truncating recessive variants suggests a null or near-null outcome for all recessive alleles.\",\n      \"method\": \"Next-generation sequencing, ophthalmic examination, retinal imaging, ERG, segregation analysis across 19 patients\",\n      \"journal\": \"JAMA network open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic genotype–phenotype correlation across 19 patients with NGS and functional ERG; multiple allele types compared\",\n      \"pmids\": [\"31199449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A truncated, labile, and mislocalized PROM1 protein results from the c.C1902G (p.Y634X) nonsense mutation associated with macular and rod-cone dystrophy, confirmed by expression in cultured cells and confocal microscopy; a second c.C1682+3A>G intronic mutation disrupts mRNA splicing (confirmed by bridge-PCR).\",\n      \"method\": \"Whole exome sequencing, transient transfection and expression in cultured cells, confocal microscopy for protein localization, bridge-PCR for splice disruption\",\n      \"journal\": \"Graefe's archive for clinical and experimental ophthalmology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based expression of mutant protein with localization readout and molecular splicing assay; two orthogonal methods, single lab\",\n      \"pmids\": [\"30588538\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PROM1/CD133 is a pentaspan transmembrane glycoprotein that selectively localizes to plasma membrane protrusions (microvilli, uropods) via interactions with PI3K and the Arp2/3 complex; its N-glycosylation state determines recognition by the AC133 antibody epitope and governs cell-surface versus intracellular distribution. Transcription is driven by tissue-specific alternative promoters regulated by DNA methylation (enforced by MBD1/2/MeCP2) and by transcription factors Sp1, Myc, and nucleolin. Intracellularly, CD133 undergoes dynein/HDAC6-dependent trafficking to the pericentrosomal recycling endosome where it sequesters GABARAP to suppress ULK1-mediated autophagy initiation, thereby maintaining an undifferentiated state and suppressing ciliogenesis. At the plasma membrane, CD133 forms a complex with E-cadherin and β-catenin and promotes Wnt/β-catenin signaling by protecting β-catenin from degradation; it also modulates microvesicle release via RhoA/Rac1 GTPases. In neurons, prominin-1 binds ALK4 to synergistically phosphorylate Smad2 and down-regulate cholesterol biosynthesis, thereby promoting axon regeneration. In the retina, loss-of-function of PROM1 causes photoreceptor degeneration (retinitis pigmentosa and cone-rod dystrophy).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PROM1/CD133 is a pentaspan transmembrane glycoprotein marking stem and progenitor populations across hematopoietic, epithelial, neural, and endothelial lineages, where it selectively concentrates in plasma-membrane protrusions and is lost upon differentiation [#1, #0, #2]. At the cell surface it shapes membrane architecture: phosphorylation of tyrosine 828 by Src-family kinases drives interactions with PI3K and the Arp2/3 complex to increase and remodel microvilli, and silencing prominin-1 abolishes uropod-associated microvilli in hematopoietic stem cells [#12]. Its detection by the AC133 antibody is governed by N-glycosylation rather than protein abundance, with complex N-glycan processing (MGAT4C) and glycosylation state determining cell-surface epitope recognition and intracellular versus surface distribution [#4, #5]. The same Y828 trafficking switch routes internalized CD133 by dynein/HDAC6-dependent transport to the pericentrosomal recycling endosome, where it sequesters GABARAP to block ULK1-mediated autophagy initiation and thereby suppresses ciliogenesis and neurite outgrowth, maintaining an undifferentiated state [#11]. CD133 reinforces stemness through signaling: it forms a complex with E-cadherin and \\u03b2-catenin that protects \\u03b2-catenin from degradation and sustains TCF/LEF transcription [#10], and in neurons binds the TGF-\\u03b2 type I receptor ALK4 to potentiate Smad2 phosphorylation and lower cholesterol biosynthesis, promoting axon regeneration [#9]. Transcription of PROM1 is driven by multiple tissue-specific alternative promoters under epigenetic control, repressed by promoter CpG methylation and methyl-CpG-binding proteins (MBD1/2, MeCP2) and activated by Sp1, Myc, nucleolin, and in MLL-rearranged leukemia by an MLL-AF4-bound intragenic enhancer [#3, #6, #8, #18, #17]. Loss-of-function PROM1 mutations cause autosomal recessive retinitis pigmentosa and cone-rod dystrophy with macular degeneration [#21, #22].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that AC133 is a novel 5-transmembrane stem-cell surface glycoprotein gave the field a defined molecular marker of long-term repopulating hematopoietic stem cells.\",\n      \"evidence\": \"cDNA cloning and flow cytometry plus in vivo fetal sheep transplantation with secondary engraftment\",\n      \"pmids\": [\"9389720\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No molecular function assigned beyond marker status\", \"Mechanism linking expression to repopulating potential unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrating that human AC133 is the orthologue of mouse prominin and selectively localizes to plasma-membrane protrusions independent of epithelial phenotype defined its conserved subcellular localization principle.\",\n      \"evidence\": \"Immunofluorescence, EM, immunoprecipitation, and ectopic expression in fibroblasts; parallel identification of CD34+VEGFR-2+ endothelial precursors losing AC133 on differentiation\",\n      \"pmids\": [\"10681530\", \"10648408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants of protrusion targeting not yet defined\", \"Functional consequence of protrusion localization unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapping multiple tissue-specific alternative promoters and showing methylation-silenced isoforms explained how a single locus achieves lineage-restricted expression.\",\n      \"evidence\": \"5'-UTR isoform cloning, luciferase reporter assays, and in vitro promoter methylation; alternative splicing producing the surface-targeted AC133-2 isoform\",\n      \"pmids\": [\"14630820\", \"12042327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which transcription factors drive each promoter not yet identified\", \"In vivo relevance of individual isoforms unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linking promoter CpG methylation state to CD133 expression in ovarian cancer and glioblastoma established epigenetic control as the switch between marker-positive and marker-negative tumor populations.\",\n      \"evidence\": \"Bisulfite sequencing, in vitro promoter methylation, and pharmacological demethylation/HDAC inhibition across sorted populations and primary tumors\",\n      \"pmids\": [\"18836486\", \"18679414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trans-acting factors reading methylation state not defined here\", \"Whether methylation changes are cause or consequence of differentiation unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showing that AC133 epitope loss reflects altered glycosylation rather than protein loss resolved a major confound in cancer-stem-cell marker interpretation.\",\n      \"evidence\": \"Cell-surface biotinylation, bacterial expression of unglycosylated CD133, and glycosylation-site mutagenesis with flow cytometry; functional differentiation suppression demonstrated in neuroblastoma\",\n      \"pmids\": [\"20068153\", \"20818439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of differential glycosylation beyond epitope masking unknown\", \"Glycosyltransferases responsible not identified in this study\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying Sp1, Myc, and methyl-CpG-binding repressors (MBD1/2, MeCP2), and later nucleolin, as direct promoter regulators connected the epigenetic state to a defined transcription-factor network.\",\n      \"evidence\": \"Gel-shift with supershift, ChIP, reporter assays with TF overexpression and pharmacological inhibition (2012); nucleolin ChIP on promoter P1 with functional HSPC readouts (2015)\",\n      \"pmids\": [\"22945648\", \"26183533\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Combinatorial logic of these factors at distinct promoters unresolved\", \"Direct nucleolin mechanism on chromatin not detailed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"A genome-wide RNAi screen establishing that N-glycan biosynthesis and MGAT4C are required for surface AC133 recognition defined the post-translational basis of epitope display.\",\n      \"evidence\": \"Pooled RNAi screen with orthogonal validation, tunicamycin treatment, and N-glycan-site mutagenesis with flow cytometry\",\n      \"pmids\": [\"21937449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How glycosylation alters trafficking versus folding not separated\", \"Physiological signal controlling glycan state unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defining Y828-phosphorylation-dependent interactions with PI3K/Arp2/3 (membrane), ALK4/Smad2 (neurons), and E-cadherin/\\u03b2-catenin (Wnt) converted CD133 from a passive marker into an active signaling and morphogenetic effector.\",\n      \"evidence\": \"Site-directed Y828 mutagenesis with Co-IP and microvillar morphology (Traffic); Co-IP plus Prom1 KO and in vivo axon regeneration (PNAS); Co-IP plus knockdown with TCF/LEF reporter (Stem Cells Transl Med)\",\n      \"pmids\": [\"30328220\", \"32554499\", \"29431914\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reciprocal validation of E-cadherin/\\u03b2-catenin complex limited to single lab\", \"How one Y828 phospho-switch coordinates divergent partner choices unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showing that dynein/HDAC6-dependent pericentrosomal trafficking lets CD133 sequester GABARAP to block ULK1 and suppress autophagy, ciliogenesis, and neurite outgrowth provided a mechanistic route by which CD133 enforces an undifferentiated state.\",\n      \"evidence\": \"Co-IP (CD133-HDAC6, CD133-GABARAP), dynein/Src inhibition, ULK1 and autophagy-flux assays, cilium and neurite readouts\",\n      \"pmids\": [\"30783186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of autophagy suppression to stemness in vivo not measured\", \"Interplay between surface signaling and endosomal pool unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing CD133 controls microvesicle release via RhoA/Rac1 and is itself driven by an MLL-AF4-bound intragenic enhancer in leukemia extended its roles to intercellular oncoprotein transfer and a defined oncogenic transcriptional circuit.\",\n      \"evidence\": \"Gain/loss-of-function with GTPase activity and KRAS-transfer assays (FASEB, 2018); ChIP-seq, Hi-C, and CRISPR/shRNA growth assays in MLL-AF4 ALL (Leukemia, 2020)\",\n      \"pmids\": [\"30521383\", \"32242051\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Microvesicle GTPase mechanism from single lab\", \"Whether enhancer dependence generalizes beyond MLL-rearranged leukemia unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying recessive null and dominant missense PROM1 mutations as causes of photoreceptor degeneration linked loss of CD133 function directly to human retinal disease.\",\n      \"evidence\": \"Linkage and segregation analysis, NGS, ERG, and mutant-protein expression with confocal localization across families and a 19-patient cohort\",\n      \"pmids\": [\"17605048\", \"31199449\", \"30588538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular role of PROM1 in photoreceptor outer-segment biology not mechanistically defined in corpus\", \"How truncated/mislocalized protein causes degeneration unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the single Y828 phospho-switch and glycosylation state are coordinated to partition CD133 between surface morphogenetic/signaling functions and the endosomal autophagy-suppressing pool, and how this governs differentiation in normal versus malignant stem cells, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating surface, signaling, and endosomal roles\", \"Structural basis of partner selection unknown\", \"Direct in vivo link from autophagy/Wnt regulation to retinal degeneration not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [9, 11, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 16]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 2, 12]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [11, 14]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3, 6, 7, 8, 17, 18]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDH1\", \"CTNNB1\", \"ACVR1B\", \"GABARAP\", \"HDAC6\", \"PIK3\", \"ARPC2\", \"NCL\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":7,"faith_pct":57.142857142857146}}