Figure 6

Downregulation of SIK3 increases the sensitivity to inhibitors of Aurora kinases and PLK1. (a) SIK3 depletion increases the sensitivity to inhibitors of Aurora kinases and PLK1. HeLa cells were transfected with control small interfering RNA (siRNA) or siSIK3. After 24 h, the cells were treated with buffer, AURKAi (MK-5108), AURKBi (Barasertib), AURKA/Bi (Alisertib), or PLK1i (BI-2536), as described in Materials and Methods. After another 24 h, the cells were harvested and analyzed with flow cytometry. (b) SIK3 depletion promotes AURKBi-induced mitotic slippage. HeLa cells expressing histone H2B-GFP were transfected with control siRNA or siSIK3. After 24 h, the cells were treated with 12.5 nM of Barasertib. After 2 h, individual cells were tracked using time-lapse microscopy for 24 h. 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) SIK3 depletion promotes PLK1i-mediated mitotic cell death. HeLa cells expressing histone H2B-GFP were transfected with control siRNA or siSIK3. After 24 h, the cells were treated with 1.25 nM of BI-2536. After 2 h, individual cells were tracked using time-lapse microscopy for 24 h. Each horizontal bar represents one cell (n=50). Key: light gray=interphase; black=mitosis (from DNA condensation to anaphase or mitotic slippage); truncated bars=cell death. (d) The onset of anaphase is delayed after inhibition of PLK1 and depletion of SIK3. HeLa/H2B-GFP cells were transfected, challenged with BI-2536, and imaged with time-lapse microscopy as described in c. The duration from prometaphase to metaphase and from metaphase to anaphase was quantified (average±90% CI). Transfection of siSIK3 significantly increased the duration of mitosis after BI-2536 challenge (P<0.05; unpaired t-test)