Figure 4.

Inhibition of aurora-B activity by AMG 900 leads to polyploidization associated with extra centrosomes, induction of apoptosis, and Bcl-2 cleavage in MOLM-13 cells. A, Cells were treated with AMG 900 at 10 nmol/L or DMSO for 48 hours. Representative confocal images of treated mitotic cells stained with p-histone H3 (red) and pericentrin (green) antibodies and DAPI (blue) (magnification x600). B, Cells treated with AMG 900 at 33 nmol/L, nutlin-3a at 2.5 μmol/L, or DMSO for 24 and 48 hours. Western blot analysis showing protein expression of total p53, MDM2, p21, cl-caspase-3, Bax, Bcl-2, Mcl-1, Bcl-xL, and β-actin. Arrows indicate specific target protein band. C and D, Cells pre-incubated ± caspase inhibitors (Z-VAV-fmk, Z-DEVD-fmk) at 10 μmol/L for 2 hours before treatment with AMG 900 at 33 nmol/L or DMSO for 48 hours. Cell count and Western blot analysis. C, Cell count represented as POC (mean ± SD). D, Protein expression profile of cl-caspase-3, Bax, Bcl-2, and β-actin. E, Cells treated with AMG 900 at 33 nmol/L or DMSO for 48 hours. Annexin-V/JC-1 analysis. Measurement of annexin-V positivity percentage (left graph, mean ± SD) and JC-1 dye red mean fluorescence intensity (MFI) for AMG 900 treated annexin-V negative and positive populations (right graph, mean ± SD).