Fig. 5

PDIA3P1 disrupts the interaction between OCT4 and the E3 ligase WWP2. A UbiBrowser 2.0 was used to analyze the E3 ligase that interacts with OCT4. B After cells were transfected with ITCH cDNA, western blot showed OCT4 protein decreased in a dose‐dependent way. C Overexpressing of WWP2 significantly suppressed OCT4 protein expression in esophageal cancer cells. D, E Cells were transfected with a control, or PDIA3P1 expression plasmid or the combination of PDIA3P1 expression plasmid and ITCH cDNA (D) or WWP2 cDNA (E). OCT4 levels were detected by western blot. F Interactions between OCT4 and WWP2 in ESCC cells were verified via Co-IP assays. G OCT4 was immunoprecipitated and immunoblotted with the indicated antibodies in KYSE-30 and KYSE-150 cells cotransfected with PDIA3P1 and WWP2. H co-IP with anti-OCT4 antibody for detecting the interaction of WWP2 in the PDIA3P1 silenced cells. I The effect of PDIA3P1 overexpression on the interaction between OCT4 and WWP2 was determined by co-immunoprecipitation assay in KYSE-30 and KYSE-150 cells. J Cells transfected with full-length or mutants Δ1 or Δ5 of PDIA3P1, respectively in ESCC. Immunoprecipitated and western blot of the ubiquitination of OCT4. K The effect of full-length or mutants Δ1 or Δ5 of PDIA3P1, respectively overexpression on the interaction between OCT4 and WWP2 was determined by co-immunoprecipitation assay in KYSE-30 and KYSE-150 cells. L KYSE-30 and KYSE-150 cell lines were transfected with OCT4 cDNA, the expression of OCT4 was analyzed by western blotting. M CCK-8 assay of the cell proliferation after simultaneous overexpression of OCT4 and WWP2 in ESCC cells (* for difference between transfected vector and OCT4 cDNA; ^# for difference between transfected OCT4 cDNA and co-transfected OCT4 cDNA and WWP2 cDNA). N EdU assays were performed to assess the proliferative ability of ESCC cells with simultaneous overexpression of OCT4 and WWP2. O, P KYSE-150 cells co-transfected with OCT4 cDNA and WWP2 cDNA, transwell assays were conducted to examine the effects of cell migration and invasion (O), and western blot shows expression levels of E-Cadherin, N-Cadherin, Vimentin and Snail (P). Q The apoptosis analysis of OCT4 and WWP2 simultaneous overexpression by flow cytometry in KYSE-150 cells. M-O and Q, all datas represent the mean ± S.D. of triplicates, for difference from the transfected with vector cells by ANOVA with Dunnett's correction for multiple comparisons. **p < 0.01, ***p < 0.001; ^##p < 0.01, ^###p < 0.001