Decitabine-induced aneuploidy and cell death is preceded by a DNA damage-induced transient G2 block. (A) Cell-cycle distribution of Akata cells 24 and 48 hours after 10 Gy of γ-IR or start of culture in the presence of 2 μM decitabine (Dec). The amount of cells with a less than diploid DNA content was regarded as apoptotic (ie, sub-G1, only number shown). (B) Decitabine and γ-IR treatment transiently arrests Akata cells in the G2 phase. To distinguish between the G2 and M phase of the cell cycle, loss of granularity due to nuclear envelope breakdown in mitosis was used during FACS analyses. To be able to set the M-phase gate correctly, Akata cells were treated with 1 μg/mL Taxol, which resulted in an accumulation of cells in gate R3. The remaining cells with the same DNA content but with higher granularity were collected in gate R2, depicted as the G2-phase. Quantifications of the cell-cycle distribution are shown in the table. (C) Decitabine treatment induces a DNA damage response (DDR). Akata cells were treated with either 5 μM decitabine or 10 Gy of γ-IR and analyzed for the presence of phosphorylated H2AX (γ-H2AX) in foci by immunofluorescence. The graph shows quantification of the percentage of cells with a signal that exceeds that of an arbitrarily chosen background staining. The results are based on quantifications of at least 100 cells counted from at least 3 different slides. A representative staining of a cell subjected to DNA damage is shown in Figure S4C. The bottom panel shows a Western blot analysis of lysates from cells treated with decitabine or γ-IR. The loading order is the same as the label on the graph. (D) Decitabine treatment leads to phosphorylation of p53 on serine 15. KemI and KemIII cells were treated with either decitabine or γ-IR and analyzed for phosphorylation of p53 serine 15. Both mutant (KemI) and wild-type (KemIII) p53 showed serine 15 phosphorylation, which is indicative of a DNA damage response.