Cyclin B1 overexpression overcomes cyclin A ^KO -mediated G ₂ -M delay. (A) Conditional gene silencing of cyclin A. HeLa cells expressing ^AIDcyclin A without endogenous cyclin A were generated (^AIDA^KOA). CDK2 was further disrupted in ^AIDA^KOA cells using CRISPR-Cas9. The cells were either left untreated or treated with DI for the indicated time for immunoblotting analysis. (B) Codepletion of cyclin A and CDK2 leads to extensive G₂/M delay. HeLa or ^AIDA^KOA cells with or without CDK2 were either left untreated or treated with DI for 24 h. Cell cycle distribution was analyzed with flow cytometry. (C) Loss of cyclin A results in cell cycle delay in both S and G₂/M. Cells were treated with DI for 24 h and pulsed with BrdU (30 min) before being analyzed with bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G₁; green: G₂/M). The percentage of cells at different cell cycle stages (excluding BrdU-negative S) was quantified (mean and SEM from three independent experiments). (D) Significant cell cycle delay in the absence of cyclin A and CDK2. Cells were preincubated with DI for 6 h to deplete cyclin A before individual cells were tracked using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown (raw data for individual cells are presented in Fig. S4 A). ****P < 0.0001. (E) Loss of cyclin A–CDK2 delays cell cycle progression. Cells were subjected to live-cell imaging analysis as described in D. Box-and-whisker plots show the elapsed time between the end of the first mitosis to the end of the second mitosis. ****P < 0.0001; ***P < 0.001; *P < 0.05. (F) Cyclin A is an essential gene in HeLa cells. ^AIDA^KOA and ^AIDA^KOA^KOCDK2 cells were cultured with or without DI. After 2 wk, the cells were fixed, stained with crystal violet, and the number of colonies was quantified. Representative images and Mean ± SEM from four independent experiments are shown. ****P < 0.0001. (G) Simultaneous cyclin A and CDK2 depletion during G₂-M. Cells were synchronized using double thymidine block, released into drug-free or DI-containing medium, and harvested at different time points for immunoblotting analysis (upper panel). DNA content was analyzed using flow cytometry (lower panel). (H) Alleviation of cyclin A^KO-induced G₂-M delay by cyclin B1 requires CDK2. A stable cell line expressing YFP-tagged cyclin B1 (B1-YFP) was established from ^AIDA^KOA cells. CDK2 was further disrupted to obtain ^AIDA^KOA^KOCDK2 cells expressing B1-YFP. Following double thymidine synchronization, cells were left untreated or treated with DI for 7 h to before analyzed using time-lapse imaging. Separate plates of cells were harvested at 3 h after the start of live-cell imaging for immunoblotting analysis to confirm protein expression. Raw data for individual cells are presented in Fig. S4 C. ****P < 0.0001; **P < 0.01. (I) Enhanced interaction between cyclin B1 and CDK2 in the absence of cyclin A. ^AIDA^KOA cells, with or without ectopically expressed cyclin B1, were synchronized in G₂ using a double thymidine block. Lysates were prepared and subjected to immunoprecipitation using antibodies against CDK1 or CDK2. The total lysates and immunoprecipitates (IP) were analyzed using immunoblotting. Note that the PSTAIRE antibody recognizes both CDK1 and CDK2. The band intensities of cyclin B1-YFP in the IP were quantified and normalized to -DI (mean ± SEM from three independent experiments). Source data are available for this figure: SourceData F7.