Conditional depletion of cyclin B induces defective mitotic entry and pre-NEBD slippage. (A) Pre-NEBD slippage in cyclin B-depleted cells. Different cell lines expressing histone H2B-GFP were synchronized using a double thymidine block and released into drug-free or DI-containing medium for 6 h to turn off cyclin B1 before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); cell death (truncated bars); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). The plot at the bottom shows the elapsed time between mitotic entry and exit (or cell death). For cells exhibiting pre-NEBD slippage, the time of mitosis was defined as from cell rounding to the appearance of cytoplasmic processes before cell flattening (blue). Mean ± SEM (n = 50). Mann–Whitney test: ****P < 0.0001; ***P < 0.001; ns P > 0.05. (B) Depletion of cyclin B1 lengthens the duration of mitosis. Live-cell imaging was performed to determine mean mitotic duration as described in A. Mean ± SEM from three independent experiments. (C) Depletion of cyclin B delays mitotic entry. Cell lines were synchronized using double-thymidine block and released into a drug-free or DI-containing medium. After 6 h, individual cells were tracked using live-cell imaging. The cumulative percentage of cells entering mitosis over time is shown. Note that DI-treated ^mAIDB1^KOB1B2 cells mainly entered pre-NEBD slippage instead of normal mitosis (shown in blue). Mean ± SEM from three independent experiments. Mann–Whitney test: ****P < 0.0001; ns P > 0.05. (D) Pre-NEBD slippage induced by cyclin B silencing. Cells were imaged following the procedure outlined in A. Representative images show ^mAIDB1^KOB1B2 cells undergoing mitosis in the presence or absence of DI. In DI-treated cells, the commencement of cell rounding and pre-NEBD slippage are indicated by the arrow and asterisk, respectively. Time: h:min. Scale bar: 10 µm. See Videos 1 and 2.