Inhibition of Aurora B kinase induces apoptosis in 2N to 4N cells. (A) Flow cytometry analysis of Annexin V binding. CD41⁺ sorted cells were cultured with or without AZD1152-HQPA for 48 hours. The plots show Hoechst 33342 staining, used to show the DNA content of cells against the level of Annexin V for each cell measured. After AZD1152-HQPA treatment the Annexin V⁺ cell population was 36% versus 12% in 2N to 4N fraction and 13% versus 8% in 8N or greater. Dot plots are from a representative experiment (n = 3). (B) AZD1152-HQPA induces caspase-3 activation in 2N to 4N population of MKs. Cells were cultured with or without AZD1152-HQPA for 48 hours. Then, CD41- and Hoechst 33342–stained cells with or without inhibitor were sorted into CD4⁺ 2N to 4N and 8N or greater. Western blot analysis showed that AZD1152-HQPA induced expression of p53 and p21 in both MK fractions. In contrast, the cleavage of caspase-3 and poly (adenosine diphosphate–ribose) polymerase were markedly induced in 2N to 4N MK-sorted cell population, whereas these cleavage forms were barely detectable in 8N or greater cells. (C) Quantitative reverse transcription polymerase chain reaction for BclxL with the use of RNA isolated from CD41⁺/2N to 4N and CD41⁺/8N or greater sorted cells. (D) The percentages of Annexin V⁺ cells in 2N to 4N and 8N or greater MKs overexpressing BclxL or in control (MIGR) with or without AZD1152-HQPA were compared. In the presence of AZD1152-HQPA, Annexin V⁺ cells were diminished in the 2N to 4N cell population overexpressing BclxL.