Conditional depletion of cyclin B induces defective mitotic entry and mitotic slippage. (A) DI treatment of control HeLa cells. HeLa cells expressing histone H2B-GFP were synchronized using a double thymidine block and released into a drug-free or DI-containing medium for 6 h before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); and cell death (truncated bars). The plots show the cumulative percentage of cells entering mitosis over time and the elapsed time between mitotic entry and exit. (B) Defective mitosis in the absence of cyclin B does not involve SAC activation. ^mAIDB1^KOB1B2 cells were cultured in a drug-free or DI-containing medium with or without the MPS1 inhibitor AZ3146. After 6 h, individual cells were tracked using live-cell imaging. Key: interphase (grey); mitosis (red); cell death (truncated bars); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). (C) Silencing of cyclin B prevents APC/C activation. ^mAIDB1^KOB1B2 cells expressing histone H2B-GFP were transfected with an mRFP APC/C biosensor plasmid. The cells were cultured in drug-free or DI-containing medium and analyzed using live-cell imaging. Representative images show normal mitosis and abnormal mitosis without APC/C activation. Time: h:min. Scale bar: 10 µm. (D) Loss of cyclin B1 alone does not abolish mitotic entry and exit. ^mAIDB1^KOB1 cells were synchronized using double thymidine block and released into a drug-free or DI-containing medium. The cells were harvested at the indicated time points for immunoblotting analysis. (E) CDK1 substrate phosphorylation in the absence of cyclin B1. Samples from D were subjected to immunoblotting using an antibody against phosphorylated CDK1 substrates (pTPxK). The positions of bands affected by cyclin B depletion are as described in Fig. 4 B. Source data are available for this figure: SourceData FS2.