Fig. 4
Ectopic overexpression of CDC6 promoted cell resistance to IR-induced apoptosis, altered the cell cycle distribution, and promoted senescence and EMT. a CNE2 cells stably overexpressing CDC6 were treated with or without IR. The apoptotic cells were stained with Annexin V-FITC/PI and detected by flow cytometry. b The cell cycle distribution was analyzed. c CNE2 cells were transiently transfected with or without CDC6 cDNA plasmid, and the cells were stained with β-galactosidase on day 5 after DNA transfection. d CDC6 and cell senescence-associated proteins such as p53, p53-pS15, p16, and p21 were detected by western blot 48, 72, 96, and 120 h after DNA transfection. e Cell morphology was observed daily after DNA transfection. f CDC6 and the EMT-associated proteins such as E-cadherin, Zeb1, and Vimentin were assessed by western blot. g, h Cell migration abilities were assessed by scratch wound healing assay. i, j Cell invasion abilities were assessed by transwell assay. k The radiosensitivity of CNE2 cells with/o CDC6 ectopic overexpression was assessed by using cell colonies formation assays when these cells were treated with IR (6 Gy). l CNE2 cells were transiently transfected with CDC6, and treated with IR (6 Gy). The senescent cells were stained with β-galactosidase assay, and the positive cells were quantified. The relative ratios of nonsenescent vs. senescent were calculated. m The clone number of epithelial or mesenchymal cells was quantified according to the morphology, and the ratios of epithelial vs. mesenchymal cells were compared in the CDC6-overexpressed CNE2 cells when treated with IR. *P < 0.05, ** P < 0.01, *** P < 0.001
