Bub3 and BuGZ levels affect mitosis. (A–C) Overexpression of Flag-BuGZ or Flag-Bub3 increased metaphase kinetochore Bub3 (A) or BubR1 (B), respectively, and the percentage of metaphase cells (C). Approximately 50 metaphase (A and B) or 100 mitotic cells (C) were counted per experiment. Bars, 5 µm. (D) Mps1 inhibition alleviated mitotic block caused by Flag-BuGZ or Flag-Bub3 overexpression. Approximately 500 cells were counted per experiment. (E and F) Bub3 and BuGZ rose and fell in HeLa (E) or tsBN2 (F) cells after release from DTB. Phospho-Histone H3 (P-H3) marks mitosis. GAPDH, loading control. Error bars indicate SEM. Student’s t test: ns, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001 from three experiments.