14-3-3σ sequesters CyclinB1 in the cytoplasm in Cdc7-depleted HeLa cells.

<p>(<b>A</b>) HeLa cells were treated with control or Cdc7-D siRNA for 24 hrs. Extracts were prepared and the immunoprecipitates with anti-CyclinB1 antibody (lanes 1 and 2) and input extracts (lanes 3 and 4; 20% of the extract used for immunoprecipitation) were analyzed by western blotting. (<b>B</b>) HeLa cells were treated with control or Cdc7-D siRNA, followed by transfection of a Flag-tagged 14-3-3σ-expressing plasmid. Extracts were prepared at 48 hrs after siRNA transfection and the immunoprecipitates with anti-Flag antibody (lanes 1 and 2) or normal (control) IgG (lanes 3 and 4) and input extracts (lanes 5 and 6; 17% of the extract used for immunoprecipitation) were analyzed by western blotting. The binding of Cdc2/CyclinB1 with 14-3-3σ increases after Cdc7 siRNA treatment, suggesting that 14-3-3σ retains CyclinB1 in the cytoplasm after Cdc7 depletion in HeLa cells. (<b>C</b> and <b>D</b>) HeLa cells were treated with Cdc7-D siRNA, 14-3-3σ and Cdc7-D siRNAs, 14-3-3σ siRNA and control siRNA for 48 hrs. (<b>C</b>) Western blot analysis of the whole cell extracts. (<b>D</b>) DNA contents of the cells in <b>C</b> were analyzed by FACS (10,000 cells for each) and the fraction of the cells in each cell cycle stage is presented. (<b>E</b>) HeLa cells expressing mKO2-CyclinB1 were treated with Cdc7-D and/or 14-3-3σ siRNA as indicated. The time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signals and its translocation into the nucleus was measured using the time lapse images, which started at 24 hrs after siRNA transfection. The P-value of the two-tailed unpaired t-test was calculated by Prism software. Co-depletion of 14-3-3σ led to a decrease in the overall CyclinB1 protein level (<b>C</b>), reduced cell death (<b>D</b>), and reduced the duration of its cytoplasmic retention (<b>E</b>).