Decitabine-induced cell death in Akata cells is caspase-dependent but can occur in the absence of induced caspase-2 activity. (A) Akata cells, or Akata cells expressing an shRNA against Puma (Figure S1) and/or were pretreated with 5 μM of the pan-caspase inhibitor Q-VD-OPH, were grown in the presence or absence of 2 μM decitabine (Dec). Apoptosis (= sub-G1), G1, G2 and aneuploidy (> 4N) were measured by FACS analysis of PI-stained cells and plotted in the table (inset) or DNA histograms. (B) Caspase-2 activity of Akata cells or Akata cells expressing an shRNA against Puma treated or untreated with decitabine was measured by a colorimetric method as measured by the cleavage of the caspase-2 substrate VDVAD-pNA into the chromophore p-nitroanilide (pNA). The specificity of the assay was ascertained by pretreating (1 hour) and culturing the cells in the presence of 20 μM of the caspase-2 inhibitor Z-VDVAD-FMK. The arbitrary units (AU) were calculated by multiplying the absorbance minus background measured at 400 nm with 100. (C) DNA histogram showing that blocking Puma and/or caspase-2 (with Z-VDVAD-FMK) results in a very small decrease in the amount of apoptosis (= sub-G1).