Chk2 is required for Aurora B–S331 phosphorylation in high nocodazole. (A) Aurora B–phospho-S331 (pS331) staining. Cells transfected with negative siRNA (control) or Chk2 siRNA (siChk2) were treated with 3.32 µM nocodazole (nocod) and MG132 for 4 h in the absence or presence of AZ3146 (AZ). (B) Aurora B localization in cells treated as in A. (C) Mitotic index analysis. Tetracycline-induced CHO cells expressing WT, S331A, or S331E Aurora B (AurB) were treated with 3.32 µM nocodazole in the absence or presence of Chk2 inhibitor II (inh II). ***, P < 0.001 compared with cells without Chk2 inhibitor. (D) Tetracycline-induced S331E Aurora B cells expressing Mad2-GFP were treated with 3.32 µM nocodazole and MG132 in the absence or presence of Chk2 inhibitor II for 4 h. (E) Mitotic index analysis. Tetracycline-induced cells expressing WT, S331A, or S331E Aurora B were treated with 3.32 µM nocodazole and AZ3146 for 8 h. Error bars show the SD from the means of three independent experiments. ***, P < 0.001. (F) Tetracycline-induced WT, S331A, or S331E Aurora B cells expressing Mad2-GFP were treated with 3.32 µM nocodazole, AZ3146, and MG132 for 4 h. 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 in A, B, D, and F show 1.7× magnification of kinetochores.