FIG 6.

CDC25C is necessary for loss of the PEA15-induced DNA damage-mediated G₂/M checkpoint defect. (A) Immunoblot analysis of total ERK1/2, PEA15, and histone H1 levels in cytosolic and nuclear fractions of HCT116 cells expressing NS or PEA15 shRNAs without or with gamma irradiation. (B) Immunoblot analysis of indicated proteins from HCT116 cells expressing NS or PEA15 shRNAs in the presence (+) or absence (−) of the MEK inhibitor U0126. (C) Autoradiograph of ³²P-labeled phospho-histone H1 (p-Histone H1) after incubation with CDK1/cyclin B purified from HCT116 cells expressing NS or PEA15 shRNAs. Immunoblots of CDK1 and cyclin B from in the inputs are also shown. (D) RT-qPCR analysis of CDC25C mRNA levels (n = 3) in HCT116 cells expressing NS or PEA15 shRNAs with or without an additional shRNA against CDC25C. Error bars indicate standard errors of the means. (E) Flow cytometry analysis to measure the G₂/M checkpoint status in HCT116 cells expressing NS, PEA15, or PEA15 and CDC25C shRNAs. Cells were analyzed at 12 h or 24 h post-gamma irradiation (p.i.). (F) Relative mitotic index after gamma irradiation of cells expressing the indicated shRNAs (n = 3). Error bars indicate standard errors of the means. **, P < 0.001. (G) Relative mitotic index after gamma irradiation of cells expressing the indicated shRNAs (n = 3). Error bars indicate standard errors of the means. **, P < 0.001.