Figure 4
Depletion of SIK3 induces defective mitotic exit. (a) SIK3 depletion delays mitotic exit. HeLa cells expressing histone H2B-GFP were transfected with control small interfering RNA (siRNA) or siSIK3. The cells then synchronized at prometaphase by blocking cells with nocodazole after they were released from a double thymidine procedure. After releasing from the nocodazole-mediated block, the cells were harvested at the indicated time points and analyzed with flow cytometry. The percentage of G2/M cells was quantified and plotted on the right (average±S.D. from three independent experiments). Mitotic exit was significantly slower in siSIK3-treated cells (P<0.01 at 2 h after release; unpaired t-test). (b) SIK3 depletion delays degradation of APC/C targets. HeLa/H2B-GFP cells were transfected and synchronized with nocodazole as described in a. Cell-free extracts were prepared and the expression of the indicated proteins was detected with immunoblotting. Equal loading of lysates was confirmed by immunoblotting for actin. The abundance of cyclin B1 was quantified with densitometry (average±S.D. from three independent experiments). Degradation of cyclin B1 was significantly slower in siSIK3-treated cells (P<0.05 at 2and 3 h after release; unpaired t-test). (c) SIK3 depletion delays mitotic exit. HeLa/H2B-GFP cells were transfected and synchronized with nocodazole as described in a. Individual cells were tracked with time-lapse microscopy to measure the time of anaphase onset. Transfection of siSIK3 significantly delayed anaphase onset (P=0.0087; log-rank (Mantel–Cox) test). (d) SIK3 depletion does not affect mitotic entry. HeLa/H2B-GFP cells were transfected with control siRNA or siSIK3. The cells were then synchronized at the G2 phase with the CDK1 inhibitor RO3306 and released into mitosis. Individual cells were tracked with time-lapse microscopy to measure the time of entry into mitosis and anaphase onset. Transfection of siSIK3 did not affect the time of mitotic entry (P>0.1) but significantly delayed anaphase onset (P<0.0001; log-rank (Mantel–Cox) test)
