Figure 3

Depletion of SIK3 increases the duration of mitosis in mouse and human cell lines. (a) Depletion of SIK3 increases the duration of mitosis in HeLa cells. HeLa/H2B-GFP cells were transfected with control small interfering RNA (siRNA), siSIK3, or siPLK1. After 24 h, the cells were analyzed with time-lapse microscopy. Individual cells were tracked for 24 h to measure the duration of mitosis (from DNA condensation to anaphase or cell death). Each horizontal bar represents the duration of mitosis for one cell (n=50). (b) siSIK3 increases the duration of mitosis in HeLa cells. HeLa/H2B-GFP cells were transfected with control siRNA or two different siSIK3. After 24 h, the cells were analyzed with time-lapse microscopy. The duration of mitosis was quantified (average±95% CI). Unpaired t-test: P<0.05 and 0.01 for siSIK3 no. 1 and siSIK3 no. 2, respectively. (c) Downregulation of SIK3 with siRNAs. HeLa/H2B-GFP cells were transfected with control siRNA or two different siSIK3. The cells were harvested after 24 h and analyzed with immunoblotting. FLAG-HA-tagged SIK3 expressed in HeLa cells was used as a standard. (d) siSIK3 increases the duration of mitosis in Hep3B cells. Hep3B/H2B-GFP cells were transfected with control siRNA or siSIK3. After 24 h, the cells were analyzed with time-lapse microscopy. The duration of mitosis was quantified (average±95% CI). (e) siSIK3 delays mitotic exit. HeLa/H2B-GFP cells were transfected with control siRNA or siSIK3. After 24 h, the cells were analyzed with time-lapse microscopy. Representative control siRNA- and siSIK3-transfected cells are shown