Fig 2: c-Kit-mediated phospho-PHBY259 in the membrane raft associates with Notch3 to regulate the Notch3—PBX1 signaling pathway. a Analysis of Notch3 (FL & NTM) and PBX1 protein expression in SKOV3_vector and SKOV3_c-Kit cells by western blotting. The quantification data represent the mean ± SD of three independent experiments. **, P < 0.01; *, P < 0.05, t test. b SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA, or mutant PHB plasmid (pD-PHBY259F-HA) for 48 h. The levels of Notch3 (FL & NTM) and PBX1 were analyzed by western blotting. c Transfection with empty vector or mutant PHB plasmid (pD-PHBY259F-HA) in SKOV3_c-Kit or SKOV3GL-G4 cells for 48 h. The levels of Notch3 (FL & NTM) and PBX1 were analyzed in whole cell lysate (WCL, top). Endo-PHBY259, PHB and exo-PHB expression was analyzed in the lipid raft domain (bottom) by western blotting. d SKOV3 cells transfected with mutant PHB plasmid (pD-PHBY259F-HA) were treated with or without chloroquine (25 µM) (or MG132; 20 µM), and the level of Notch3 was determined by western blotting and compared to that in SKOV3 cells transfected with wild-type pD-PHB-HA plasmid. e To monitor protein stability, SKOV3 cells transfected with wild-type pD-PHB-HA or mutant PHB plasmid (pD-PHBY259F-HA) were treated with cycloheximide (CHX; 50 µM), and the level of Notch3 was measured at the indicated time points. Data represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test. f SKOV3_c-Kit cells were transiently transfected with plasmid pD-PHB-HA, and immunoprecipitation was performed with anti-HA tag antibody. Coimmunoprecipitation (Co-IP) proteins were detected by western blotting with anti-c-Kit, anti-PHB, and anti-Notch3 antibodies. g Localization of c-Kit, raft-PHB, and Notch3 was analyzed in SKOV3_c-Kit cells through confocal microscopy. Scale bar = 10 µm. h SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA, or mutant PHB plasmid (pD-PHBY259F-HA) for 48 h. PHB was immunoprecipitated with anti-HA antibody, and coimmunoprecipitation of Notch3 was detected by western blotting. Blots are representative of three independent experiments

Fig 3: Phosphorylated PHB at Y259 by c-Kit in the membrane raft upregulates ß-catenin—ABCG2 signaling. a Analysis of ß-catenin and ABCG2 protein expression in SKOV3 and SKOV3_c-Kit cells by western blotting. b SKOV3 cells were transfected with empty vector, wild-type pD-PHB-HA, or mutant PHB plasmid (pD-PHBY259F-HA) for 48 h. The levels of ß-catenin and ABCG2 were analyzed by western blotting. c Transfection with empty vector or mutant PHB plasmid (pD-PHBY259F-HA) in SKOV3_c-Kit, SKOV3GL-G4 or KURAMOCHI cells for 48 h. The levels of ß-catenin and ABCG2 were analyzed by western blotting. Blots are representative of three independent experiments

Fig 4: Phospho-PHBY259 in the membrane raft promotes ovarian cancer stem cell survival and potentiates drug-resistant phenotypes. a SKOV3_c-Kit and SKOV3GL-G4 cells were transfected with empty vector or mutant PHB plasmid (pD-PHBY259F-HA) for 48 h and plated at a density of 300 cells/well in 6-well plates for 14 days. The colony formation was visualized with crystal violet. b Spheroid-forming assay in SKOV3_c-Kit (or SKOV3GL-G4) cells transfected with empty vector or pD-PHBY259F-HA plasmid. Representative photos were taken on days 4, 8, and 12. Spheroid sizes were digitally determined. c Carboplatin sensitivity assay. SKOV3 cells were transfected with empty vector or wild-type pD-PHB-HA for 48 h. Cells were treated with carboplatin for 72 h, and cell viability was detected. d SKOV3_c-Kit and SKOV3GL-G4 cells were transfected with empty vector or pD-PHBY259F-HA plasmid. After treatment with carboplatin for 72 h, cell viability was measured using the CCK-8 Assay Kit. The above data represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test

Fig 5: c-Kit phosphorylates PHB at tyrosine 259. a Different amounts of active c-Kit kinase domain (0, 20, 40, 80, 160 ng) reacted with 1 µg PHB-His in an in vitro kinase assay. The luminescence was read using a Biotek microplate reader. Data represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test. b Activity of c-Kit kinase, phospho-PHBY259-His and PHB-His from the in vitro kinase assay was analyzed by western blotting. For preabsorption control, anti-phospho-PHBY259 antibody was pre-incubated with an excess amount of the phosphopeptide spanning phospho-tyrosine 259 of PHB (IAYQLSRSRNIT (pY)LPAGQSVLLQ; 10 µg peptide/1 µg antibody) for 2 h at room temperature and then used for western blotting in the rightmost lane. Blots are representative of three independent experiments