Fig. 3.

CDK1 activity is critical for maintaining the mitotic state. (A) Nocodazole-arrested cells rapidly exit mitosis in the presence of RO-3306. HeLa cells arrested in prometaphase by nocodazole treatment (16 h) were followed by time-lapse photography after the addition of 9 μM RO-3306. (B) Inhibition of CDK1 activity in nocodazole-arrested mitotic cells triggers chromosome decondensation and nuclei reformation. Purified population of nocodazole-arrested metaphase cells was collected by shake-off and treated with RO-3306 (9 μM). Mitotic spreads were prepared at different times and stained with Giemsa. (C) HeLa cells were treated as above but stained for DNA with Hoechst 33258. (D) Western blot analysis of phospho-H3 and phospho-pRB during RO-3306-induced exit from mitosis. (E) Inhibition of cyclin B1 degradation does not prevent mitotic exit in RO-3306-treated HeLa cells. Purified nocodazole-arrested metaphase cells were treated with solvent (a and b) or 10 μM MG132 (c and d) for 30 min followed by the addition of 9 μM RO-3306 for 2 h (b and d). (F) Western blot analysis of cyclin B1 and phospho-H3 in the cells treated as above. (Scale bars: 20 μm.)