Figure 5.

Downregulation of Aurora A recapitulates the effects of MK8745. (A) Aurora A was inhibited in HCT116 and its p53^−/− cell lines with 5 µM MK (MK) or by downregulating with specific siRNA sequence (ASi) and treatment with with 5 µM MK (ASi+MK), or by downregulating Aurora B with specific siRNA sequence (BSi) along with control siRNA (CSi) and flow cytometry analysis was performed after staining with propidium iodide. (B) HCT 116 and p53^−/− cells were treated with (MK, 5 µM) for 24 h was compared with 48 h of control (CSi) or Aurora A siRNA (ASi) and nuclei were visualized after staining with DAPI. (C) HCT116 cells were treated with 5 µM MK or siRNA specific for Aurora A for 48 h and apoptosis was measured by FACScan analysis after probing for Annexin (gray bar), after staining with Propidium iodide for <2N DNA (white bar) or quantitated using microscopy after staining with DAPI (black bar). (D) (i and iii) HCT 116 and p53^−/− cells were treated with (MK, 5 µM) for 24 h was compared with 48 h of control (CSi) or Aurora A siRNA (ASi) and western blot analysis was performed for phospho p53 (ser15), p53, p21, cleaved PARP and cleaved caspase3. Aurora A downregulation is shown and cytochrome C release to cytosol. HCT 116 cells were either transfected with CSi, ASi or BSi were treated with vehicle, MK or ABI (AZD 100 nM) and western blot analyses were performed for Aurora A, phospho Aurora A, Aurora B, cleaved PARP, phospho Histone H3 (ser10) and tubulin. All results are representative of 3–4 independent experiments.