Figure 3

Cdc20 regulates condensin II function by degradation of CAP-H2 during mitosis. (A) HeLa cells were synchronized at mitosis by nocodazole with or without BI2536, and then the cells were treated with MG132. Cell lysates were analyzed by immunoblotting with indicating antibodies. A ratio of CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of CAP-H2/Tubulin in untreated cells was defined as 1.0. (B) HeLa cells were transfected with non-target siRNA, β-TrCP1/2 siRNA, Cdc20 siRNA or Cdh1 siRNA. At 24 h after transfection, cells were treated with nocodazole and BI2536 for 16 h. Mitotic cell lysates were immunoblotted with the indicated antibodies. A ratio of CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of CAP-H2/Tubulin in each of siRNA transfected and nocodazole treated cells was defined as 1.0. (C) HeLa cells were transfected with Flag-vector or Flag-Cdc20. At 24 h after transfection, cells were treated with nocodazole and BI2536 for 16 h. Mitotic cell lysates were immunoblotted with the indicated antibodies. A ratio of CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of CAP-H2/Tubulin in Flag-vector transfected and nocodazole treated cells was defined as 1.0. (D) Fixed HeLa cells were stained with anti-phospho-histone H3-Ser10. DNA was stained by DAPI. Degrees of prophase chromosome condensation were defined in the two categories and the representative cells are shown. Scale bar, 10 μm. (E) HeLa cells were transfected with Flag-vector or Flag-Cdc20 and synchronized at G1/S phase by thymidine treatment. Cells were then released into the cell cycle. At 9 h after release, cells were fixed and were stained with anti-phospho-histone H3-Ser10 and anti-Flag. The percentage of each category, defined in D, was calculated. n > 100 cells per indicated condition were analyzed. Data represent mean ± SD from three independent experiments. Statistical analysis was performed with the Student’s t test (*P < 0.05). (F) The cells were transfected and synchronized as described in E. Cell lysates were analyzed by immunoblot analysis using indicated antibodies. A ratio of CAP-H2/Tubulin was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of CAP-H2/Tubulin in Flag-vector transfected cells was defined as 1.0.