Myc provokes G2 checkpoint override that sensitizes p53 knockout mouse embryo fibroblasts (MEFs) to decitabine-induced cell death. (A) Low passage p53^−/− MEFs were infected with retroviruses made either with MSCV-IRES-puro (vector control) or with MSCV-MycER-IRES-puro vectors. After selection, the cells were cultured in the presence or absence of 4-HT, to induce nuclear translocation and activation of MycER, and either treated with 10 Gy γ-IR or 2 μM decitabine (Dec) for the indicated time points. The cells were harvested and stained with propidium iodide and their cell-cycle distribution was analyzed by FACS. (B) The same cells as in panel A were also seeded into 6-well plates and cultured for 10 days after γ-IR or decitabine. Cultures with freshly added decitabine daily for 3 or 10 days produced the same results and a representative experiment of 3 independent clonogenic survival assays is shown. (C) Western blot analysis showing levels of total and phosphorylated cyclin-dependent kinase 1 (Cdc2) in p53^−/− MEFs subjected to decitabine and Myc activation for the indicated times. Less phosphorylated Cdc2 suggests checkpoint override. (D) Akata cells were treated with either decitabine or γ-IR and analyzed for phosphorylation of Cdc2 by Western blot analysis.