Figure 7

Downregulation of SIK3 increases the sensitivity to antimitotic drugs. (a) SIK3 depletion enhances the sensitivity to the Eg5 inhibitor (Eg5i) SB743921. HeLa cells were transfected with control small interfering RNA (siRNA) or siSIK3. After 24 h, the cells were treated with buffer or 1.25 nM of SB743921. After another 24 h, the cells were harvested, fixed, and analyzed with flow cytometry. (b) SIK3 depletion promotes SB743921-mediated mitotic arrest. 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 SB743921. 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. (c) Early mitotic delay after inhibition of Eg5 and depletion of SIK3. HeLa/H2B-GFP cells were transfected, challenged with SB743921, and imaged with time-lapse microscopy as described in b. The duration from prometaphase to metaphase and from metaphase to anaphase was quantified (average±90% CI). Transfection of siSIK3 significantly increased prometaphase delay after SB743921 challenge (P<0.0001; unpaired t-test). (d) SIK3 depletion enhances the sensitivity to SB743921 in drug-resistant cells. HeLa cells and two independent clones of SB743921-resistant cells (SBR no. 1 and SBR no. 4) were transfected with control siRNA or siSIK3. After 24 h, the cells were treated with buffer or 10 nM of SB743921. After another 24 h, the cells were harvested, fixed, and analyzed with flow cytometry. (e) SIK3 depletion does not enhance the mitotic defects induced by the HDAC inhibitor SAHA. HeLa cells expressing histone H2B-GFP were transfected with control siRNA or siSIK3. After 24 h, the cells were treated with 1 nM of SAHA. After 2 h, individual cells were tracked using time-lapse microscopy for 24 h. The duration from prometaphase to metaphase and from metaphase to anaphase was quantified (n=50, average±90% CI). There is no significant additive effects of the two treatments (P>0.1; unpaired t-test)