Delayed mitotic entry in the absence of cyclin A and CDK2. (A) Mitotic entry is significantly delayed in ^AIDA^KOA and ^AIDA^KOA^KOCDK2 cells. Cells were pre-incubated with DI for 6 h to deplete cyclin A, followed by live-cell imaging for 48 h (n = 50). Key: interphase (grey); mitosis (red); and cell death (truncated bars). (B) Minimal delay in S phase progression upon cyclin A silencing. ^AIDA^KOA cells were synchronized at S phase with double thymidine block and treated with DI at the second thymidine release to deplete ^AIDcyclin A. The cells were pulsed with BrdU for 30 min before harvested at each time points for immunoblotting analysis. The band intensity of ^AIDcyclin A was quantified and normalized to -DI control at t = 0 h. BrdU incorporation and DNA content were examined with bivariate flow cytometry (red: BrdU-positive; yellow: BrdU-negative S; blue: G₁; green: G₂/M). The percentages of S (BrdU-positive) and G₂ cells were quantified (mean ± SEM from four independent experiments). (C) Alleviation of cyclin A^KO-induced G₂-M delay by cyclin B1. ^AIDA^KOA and ^AIDA^KOA^KOCDK2 cells overexpressing cyclin B1 synchronized with double thymidine block were left untreated or treated with DI for 7 h to turn off cyclin A before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); and cell death (truncated bars). (D) Conditional gene silencing of cyclin A in H1299 cells. HeLa and H1299 cells expressing ^AIDcyclin A without endogenous cyclin A were left untreated or treated with DI for 24 h before immunoblotting analysis. Lysates from HeLa and H1299 cells were included as controls for the relative expression of ^AIDcyclin A and endogenous cyclin A. (E) Cyclin A depletion induces G₂-M delay in both HeLa and H1299 cells. ^AIDA^KOA cells were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 6 h, mitotic entry was analyzed using live-cell imaging (left panel; time indicates the duration after thymidine release). Raw data for individual cells are presented in the right panel. Key: interphase (grey); mitosis (red); and cell death (truncated bars). (F) Presence of cyclin B1–CDK2 complexes during normal G₂. HeLa cells were synchronized at S phase with double thymidine block. After release into fresh medium for 5 h, NOC was added to prevent mitotic exit. After 4 h, mitotic cells were removed by washing, and the attached G₂ cells were harvested for immunoprecipitation using antibodies against CDK1, CDK2, or cyclin A. Protein expression in the total lysates and immunoprecipitates (IP) was detected using immunoblotting. A negative control (no antibody was added) was included to assess the specificity of the immunoprecipitation. Source data are available for this figure: SourceData FS4.