Mitotic defects caused by cyclin B deficiency can be compensated by cyclin A overexpression. (A) Ectopic expression of cyclin A in cyclin B-deficient cells. ^mAIDB1^KOB1B2 cells were transfected with ^mRFPcyclin A expression plasmids. Cells with varying levels of ^mRFPcyclin A were sorted by flow cytometry. The cell lines were left untreated or treated with DI for the indicated time before analyzed with immunoblotting. (B) Rescue of cyclin B deficiency-induced G₂/M arrest by cyclin A overexpression. Parental and ^mAIDB1^KOB1B2 cells expressing low or high levels of ^mRFPcyclin A were treated with DI to turn off ^mAIDcyclin B1. The cells were harvested at the indicated time points and analyzed using flow cytometry. (C) Cyclin A rescues clonogenic survival in cyclin B-deficient cells. ^mAIDB1^KOB1B2 cells expressing high levels of ^mRFPcyclin A were cultured with or without DI for 2 wk. Colonies were fixed and stained. Mean ± SEM from six independent experiments. Mann–Whitney test: **P < 0.01; *P < 0.05. Note that the same data for the ^mAIDB1^KOB1B2 control cells as in Fig. 1 F were used for comparison. (D) Cyclin B deficiency-induced pre-NEBD slippage can be overcome by cyclin A overexpression. Cells expressing histone H2B-GFP were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 6 h, individual cells were tracked using live-cell imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); cell death (truncated bars); interphase after cytokinesis failure (purple); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). The plot shows the percentage of defective mitosis (mitotic slippage and cytokinesis failure). Mean ± SEM from three independent experiments. (E) Overexpression of cyclin A overcomes mitotic entry delay in cyclin B-deficient cells. Cell lines were synchronized, released into drug-free or DI-containing medium, and analyzed with live-cell imaging as described in D. The cumulative percentage of cells entering mitosis (both normal and pre-NEBD mitosis) over time is shown. Mean ± SEM from three independent experiments. Mann–Whitney test: ****P < 0.0001; ***P < 0.001; ns P > 0.05. Note that the same graph from Fig. 2 C is included for clarity for control ^mAIDB1^KOB1B2 cells. (F) Ectopic expression of cyclin A restores mitosis in cyclin B-deficient cells. Parental and cyclin A-overexpressing ^mAIDB1^KOB1B2 cells were synchronized using a double thymidine block. The cells were released into a drug-free or DI-containing medium and harvested at different time points. Protein expression was analyzed with immunoblotting. The positions of pTPxK bands affected by cyclin B depletion are indicated as described in Fig. 4 B. (G) Increased cyclin A–CDK1/2 complexes in the absence of cyclin B. Parental and cyclin A-overexpressing ^mAIDB1^KOB1B2 cells were grown in drug-free or DI-containing medium for 24 h. Lysates were prepared and subjected to immunoprecipitation using an antibody against cyclin A. Both total lysates and immunoprecipitates (IP) were analyzed with immunoblotting. (H) Increased binding of both endogenous and ^mRFPcyclin A to CDK1 upon the loss of cyclin B. Parental and cyclin A-overexpressing ^mAIDB1^KOB1B2 cells were synchronized using double thymidine block as described in F. Lysates were prepared and subjected to immunoprecipitation using an antibody against CDK1. Source data are available for this figure: SourceData F6.