Figure 1. Activation of the G₂ DNA damage checkpoint is independent on MASTL.

(A) MASTL does not vary significantly during the cell cycle. HeLa cells were synchronized using a double thymidine procedure. At different time points, the cells were harvested and analyzed with flow cytometry (upper panel) and immunoblotting (lower panel). Cyclin B1 and histone H3^Ser10 phosphorylation indicated the timing of mitosis. (B) MASTL in G₂ and mitosis. Lysates from G₂ and mitosis (M) were analyzed with immunoblotting. MASTL was extensively phosphorylated during mitosis. Cells transfected with MASTL siRNA (siMASTL) or plasmids expressing FLAG-MASTL were loaded to validate the identity of the MASTL band. (C) Low kinase activity of MASTL during interphase. Different lysates were subjected to immunoprecipitation using either control or MASTL antiserum. The kinase activity was assayed using purified 6xHis-ENSA as a substrate. (D) Downregulation of MASTL does not affect unperturbed cell cycle distribution. Transfected HeLa cells were harvested at the indicated time points and analyzed. (E) IR-mediated interphase arrest is independent on MASTL. HeLa cells were transfected as indicated. After 48 h, the cells were treated with IR (15 Gy) or incubated with nocodazole (NOC). After another 16 h, the cells were harvested for immunoblotting. (F) IR-induced G₂ arrest is independent on MASTL. HeLa cells transfected as indicated were irradiated (15 Gy). After another 16 h, the cells were analyzed with flow cytometry. NOC treatment (16 h) was used as a mitotic control. (G) MASTL does not affect IR-mediated 53BP1 foci formation or repair. HeLa cells transfected as indicated were irradiated (2 Gy) and fixed at different time points. The number of 53BP1 foci per cell was quantified (n = 100; mean ± 95% CI). Examples of 53BP1 staining of siMASTL-transfected cells in the presence and absence of IR treatment (3 h) are shown. (H) Mitosis is inhibited by IR in the absence of MASTL. HeLa cells expressing histone H2B-GFP were transfected as indicated. After 48 h, the cells were either mock-treated or irradiated (15 Gy). Individual cells were then tracked with time-lapse microscopy. Each horizontal bar represents one cell (n = 50). Grey: interphase; black: mitosis (from DNA condensation to anaphase); truncated bars: cell death.