Alisertib inhibits AURKA and AURKB in a concentration-dependent manner. (a) Alisertib induces G2/M delay or genome reduplication. HeLa cells were exposed to buffer or the indicated concentrations of Alisertib. After 24 h, the cells were harvested and analyzed with flow cytometry. The positions of 2N, 4N and 8N DNA contents are indicated. (b) Alisertib delays mitotic exit or induces slippage. HeLa cells stably expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of Alisertib. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n=50). Key: light gray=interphase; black=mitosis (from DNA condensation to anaphase or mitotic slippage); dark gray=interphase after mitotic slippage; truncated bars=cell death. (c) Different concentrations of Alisertib are involved in delaying mitotic exit and inducing slippage. Live-cell imaging of cells treated with Alisertib was described in panel (b). The duration of mitosis (mean±90% confidence interval) and the percentage of cells that underwent mitotic slippage during the imaging period was quantified. (d) Alisertib promotes apoptosis in a concentration-dependent manner. HeLa cells were incubated with the indicated concentrations of Alisertib for 48 h. The cells were then harvested and analyzed with flow cytometry. (e) Concentration-dependent cytotoxicity of Alisertib. HeLa cells were cultured in the presence of the indicated concentrations of Alisertib for 48 h. The number of live and dead cells was analyzed with trypan blue exclusion assay. (f) Concentration-dependent suppression of long-term survival by Alisertib. HeLa cells were seeded on 60-mm culture plates and grown in the presence of 250 nM or 1 μM of Alisertib. After 24 h, the cells were washed gently and propagated in normal medium for another 10–12 days. Colonies were fixed and stained with crystal violet solution (examples of the plates are shown). Average±s.d. from three independent experiments. (g) Both AURKA and AURKB are inhibited by Alisertib. Mitotic HeLa cells were obtained by exposure to nocodazole for 16 h followed by mechanical shake off. The cells were incubated with the indicated concentrations of Alisertib for 2 h. Lysates were then prepared and activated phospho-AURKAThr288 and AURKBThr232 were detected with immunoblotting. The asterisk indicates the position of an AURKB-like protein (the same throughout this study). Uniform loading was confirmed by immunoblotting for actin. In this assay, nocodazole and MG132 (a proteasome inhibitor) were added to prevent the cells from exiting mitosis. Accordingly, the total AURKA and AURKB levels remained constant throughout the experiment. (h) Alisertib prevents activation of AURKA and AURKB. HeLa cells were incubated with the indicated concentrations of Alisertib for 8 h. Nocodazole was then added for another 6 h to trap cells that entered mitosis. Lysates were prepared and analyzed with immunoblotting. Actin analysis was included to assess loading and transfer.