Skip to main content
. 2016 Feb 29;6:22230. doi: 10.1038/srep22230

Figure 2. DNA damage-induced G2 arrest can be interrupted by MASTL.

Figure 2

(A) Ectopic expression of MASTL. A HeLa cell line expressing FLAG-EGFP-MASTL was generated. Lysates were prepared and different dilutions were loaded to compare with normal HeLa cells. (B) Overexpression of MASTL does not affect checkpoint activation. HeLa cells expressing FLAG-EGFP-MASTL were irradiated (15 Gy). NOC was added to trap cells in mitosis. After 6 h, the cells were harvested and analyzed. The expression of exogenous MASTL was detected with antibodies against FLAG. (C) Expression of MASTLK72M. Cell lines expressing FLAG-EGFP-MASTLK72M under the control of doxycycline (Dox) were generated. MASTLK72M expression in different clones was compared to the cell line expressing wild type MASTL. Clone#15 was used in other experiments in this paper. (D) MASTLK72M promotes mitotic entry after DNA damage. Cells were grown in the absence or presence of Dox for 48 h to turn on or off MASTLK72M respectively before treated with IR (15 Gy). After 16 h, the cells were incubated with NOC and harvested at different time points for immunoblotting. (E) MASTLK72M accelerates mitotic entry after DNA damage. Cells were grown in the absence or presence of Dox for 48 h. The cells were then transfected with a plasmid expressing histone H2B-mRFP. After 24 h, the cells were treated with IR (15 Gy). After another 16 h, individual cells were tracked with time-lapse microscopy to analyze the time of mitotic entry (n = 50). The median mitotic entry time was plotted as a box-and-whisker chart. Expression of MASTLK72M significantly accelerated entry into mitosis (P < 0.001). (F) MASTLK72M shortens G2 arrest after DNA damage. Cells were grown in the absence or presence of Dox for 48 h. After treating with IR (15 Gy), the cells were harvested at the indicated time points and analyzed with flow cytometry. (G) MASTLK72M does not affect inactivation of the checkpoint. Cells were grown in the absence or presence of Dox for 48 h. After treating with IR (15 Gy) and NOC, the cells were harvested at the indicated time points. The expression of the indicated proteins was detected with immunoblotting.