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. 2016 Feb 29;6:22230. doi: 10.1038/srep22230

Figure 3. MASTL controls the timing of mitotic entry after DNA damage.

Figure 3

(A) Inhibition of CHK1/CHK2 bypasses the G2 DNA damage checkpoint. DNA damage activates CHK1/CHK2. This results in the activation of WEE1 and inhibition of CDC25, thereby keeping CDK1 in an inhibitory phosphorylated state. Inhibitors of CHK1/CHK2 induce damaged cells to enter mitosis. (B) MASTLK72M promotes mitotic entry after AZD7762-mediated checkpoint abrogation. HeLa cells expressing FLAG-EGFP-MASTLK72M were grown in the presence or absence of Dox(48 h) before irradiated (15 Gy). After 16 h, the cells were incubated with AZD7762 (CHK1i) and NOC (to trap cells in mitosis) and harvested at different time points. Phosphorylation of MASTL during mitosis induced a gel mobility shift (MASTL-p). (C) MASTLK72M accelerates mitotic entry after checkpoint abrogation. HeLa cells expressing FLAG-EGFP-MASTLK72M were grown with or without Dox(48 h) before transfected with histone H2B-mRFP-expressing plasmids. After 24 h, the cells were irradiated (15 Gy). After another 16 h, the cells were incubated with CHK1i. Individual cells were then tracked with time-lapse microscopy to analyze the time of mitotic entry (n = 50)(upper panel). The median mitotic entry time was plotted as a box-and-whisker chart (lower panel). MASTLK72M significantly accelerated mitotic entry (P < 0.001). (D) Depletion of MASTL delays mitotic entry after DNA damage. HeLa cells were transfected with either control or siMASTL for 48 h before irradiated (15 Gy). After 16 h, the cells were incubated with CHK1i and NOC and harvested at different time points. (E) Depletion of MASTL delays mitotic entry after checkpoint abrogation. HeLa cells expressing histone H2B-GFP were transfected with either control or siMASTL for 48 h before irradiated (15 Gy). After another 16 h, the cells were incubated with CHK1i. Individual cells were then analyzed with time-lapse microscopy as in panel (C). Depletion of MASTL significantly delayed entry into mitosis (P < 0.05). (F) Depletion of ARPP19 or ENSA delays mitotic entry after checkpoint abrogation. HeLa cells expressing histone H2B-GFP were transfected with control, siARPP19, or siENSA for 48 h before irradiated (15 Gy). After another 16 h, the cells were incubated with CHK1i and analyzed with time-lapse microscopy as in panel (C). Depletion of ARPP19 or ENSA significantly delayed entry into mitosis (P < 0.001).