Treatment with AZ3146 exacerbates mitotic exit in Chk2-deficient cells. (A) Western blot analysis of total phosphorylated Y15 (pY15), Cdk1, and actin. Cells were treated with 3.32 µM nocodazole (nocod) in the absence (control) or presence of Chk2 inhibitor II (inh II) or AZ3146 (AZ). Values at 0 h were taken as 1. (B) Cells were treated as in A and transfected with AF or WT Cdk1-GFP. Mitotic index shows the percentage of mitotic green cells/total green cells. Error bars show the SD from the means of three independent experiments. ***, P < 0.001. (C) Localization of BubR1. Cells transfected with negative siRNA (control) or Chk2 siRNA (siChk2) were treated with 3.32 µM nocodazole and MG132 for 4 h in the absence or presence of AZ3146. (D) Cells expressing Mad2-GFP were treated as in C. (C and D) Boxed values show mean green/red fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Bars, 5 µm. The insets show 1.7× magnification of kinetochores. (E) Mitotic index analysis. Cells treated with 3.32 µM nocodazole and AZ3146 for 8 h in the absence (control) or presence of Chk2 inhibitor II. Error bars show the SD from the means of three independent experiments. ***, P < 0.001. (F) Cdk1-associated histone H1 kinase activity and Western blot analysis of immunoprecipitated (IP) Cdk1 in cells treated as in E. Phosphorylated H1 (pH1) values at first lane were taken as 1.
