Figure 4
Knockdown of CDK1 induces the activation of γH2.AX and downregulation of CHK2 expression in hESC. (a) Confocal microscopy analysis showing the presence of γH2 A.X foci in hESC at 1, 2 and 3 days post transfection of control and CDK1 siRNAs. Phosphorylated histone H2A.X (γ -H2A.X foci) is shown as white dots. Chromatin is stained with DAPI (blue). Scale bar=10 μm, D=day. Images are representative of at least three independent experiments. Percentage of γ-H2A.X-positive cells is shown at the bottom. (b) Graphic representation of the average number of γH2A.X foci per nucleus in hESC during 3-day time course post CDK1 and control siRNAs transfections. Data areshown as average±S.E.M., n=3. (c) Flow cytometric analysis of γ-H2A.X on the second day post transfection and 16 h after administration of IR. Data are shown as average±S.E.M., n=3. (d) Representative images of four repeats of western blot analysis for CHK1 and CHK2 expression up to 96 h post transfection of siRNAS. β-Actin was used as the loading control. (e) Impacts of CDK1 inhibition on the regulation of key factors involved in G2 checkpoint activation analysed by western blotting at day 2 post transfection and after 6 h post IR on the same day (shown in the figure as IR group). β-Actin served as the loading control. The data represent at least three independent experiments. (f) Representative flow cytometric histograms at 2 days post transfection+6 h post IR. The percentage of cells in each stage of cell cycle was calculated using ModFit. Graphic representation of these data is shown on the right hand panel. Results are presented as mean±S.E.M. (n=3). T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05
