Cyclin A drives residual mitotic activity in the absence of cyclin B. (A) Enhanced formation of cyclin A–CDK1 complexes without cyclin B. ^mAIDB1^KOB1B2 cells synchronized with double thymidine block were released into a drug-free or DI-containing medium and harvested at different time points. Lysates were prepared and subjected to immunoprecipitation with a CDK1 antibody. Both total lysates and immunoprecipitates (IP) were analyzed with immunoblotting. (B) Contribution of cyclin A to CDK1 substrate phosphorylation in the absence of cyclin B. ^mAIDB1^KOB1B2 cells transfected with control siRNA (siControl) or siRNA targeting cyclin A (siCyclin A) were synchronized using double thymidine block. The cells were released into a drug-free or DI-containing medium and harvested at different time points for immunoblotting. The positions of pTPxK bands affected by cyclin B depletion are indicated as described in Fig. 4 B. (C) Depletion of cyclin A does not affect cyclin B expression. Cell lines transfected with control siRNA or siRNA targeting cyclin A were left untreated or treated with DI for 24 h. Lysates were prepared and analyzed with immunoblotting. (D) Suppression of mitotic entry in cyclin B-deficient cells upon cyclin A knockdown. Cell lines transfected with control siRNA or siRNA targeting cyclin A were analyzed using live-cell imaging after treatment with DI. The cumulative percentage of cells entering mitosis over time is shown. Note that DI-treated ^mAIDB1^KOB1B2 cells exhibited pre-NEBD slippage (*) instead of normal mitosis. Source data are available for this figure: SourceData F5.