Fig. 6. Activation of a replication-stress checkpoint and analysis of apoptosis and ssDNA levels. (A) Protein expression of the cell-cycle regulators Chk1-P, Chk2-P, Cdc25A, p21 and Cdk2 in whole cell extracts prepared at 96 hours from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells. Actin was used as loading control. Extracts from cells treated with the drugs hydroxyurea (10 mM) and nocodazole (100 ng/µl) were used as positive controls for the activational phosphorylation of Chk1 and Chk2. (B) Quantification of the protein levels of the cell-cycle regulators shown in A. Values represent the average of three experiments and the error bars correspond to 1 s.d. Chk1-P levels were not included in the bar graph because no detectable levels were observed. (C) Protein expression of the Cdk2 regulators Cdc25A, p21 and Cdk2 in whole cell extracts prepared after Ku80-siRNA transfection in HCT-116 Ku80^+/– cells. (D) Genomic DNA was isolated from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells after 96 hours of Ku80 silencing and was subjected to electrophoresis in order to determine its integrity. Extracts from cells treated with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), which is known to induce apoptosis, were used as positive controls. (E) Representative western blot analysis of the protein levels of full-length PARP-1 and the full-length precursor form of caspase 3, which are cleaved during apoptosis. (F) Chromatin association of the ssDNA-binding protein RPA70 at 96 hours post-transfection.