Figure 2.

Cdk1 is required for HPV E7 to abrogate DNA damage-induced G1 arrest. (A) Expression of Cdks in E7-expressing cells. The steady-state levels of Cdk1 and Cdk2 in E7-expressing or vector-containing NIKS and RPE1 cells treated with bleomycin or PBS were examined by immunoblotting. Actin-β was used as a loading control. (B) siRNAs targeting Cdk1 or Cdk2 specifically reduced their expressions. RPE1-E7 cells were transfected with Cdk1-siRNA and Cdk2-siRNA respectively. Cdk1 and Cdk2 protein levels were assessed by immunoblot analysis. Tubulin-β was used as a loading control. (C) RPE1-E7 cells were transfected with either Cdk1 or Cdk2 siRNAs and then treated with bleomycin. Cells were harvested at 72 hours post-transfection, stained with PI and analyzed by flow cytometry. A representative experiment of 6 is shown (Left panel). The percentage of cells at G1 phase is gated. Data of 6 experiments from RPE1-E7 cells are summarized (Right panel). (D) RPE1 E7 cells were transfected with either Cdk1 or Cdk2-siRNAs. Sixteen hours later, cells were treated with bleomycin for 32hrs. Cells were labeled with BrdU before being collected and stained with anti-BrdU antibody, counterstained with PI, analyzed by flow cytometry. Data from a representative of 4 experiments are shown (Left panel) and the percentage of BrdU-positive cells is gated. Data are summarized (Right panel). Error bars reflect the standard deviations of the mean. Si-NS, non-silencing siRNA.