Figure 4

RNAi against CSB in HCT116p53-/- cells. (A) The effectiveness of siRNAs against CSB in HCT116, HCT116 + chr3 and HCT116p53-/- cells was assessed by immunoblot analysis of nuclear lysates from cells that were either mock-transfected (M) or transfected with the indicated siRNA (non-targeting control (NT) or CSB). (B) HCR of a UV-damaged reporter gene was determined in non-targeting siRNA (NT) and CSB siRNA (CSB) transfected HCT116p53-/- cells. (C) The ability of mock-, NT- and CSB-siRNA transfected cells to recover nascent RNA synthesis following UV exposure was assessed in HCT116p53-/- cells. (D) The sensitivity of HCT116p53-/- cells (closed symbols, solid lines) transfected with NT (triangles) or CSB (inverted triangles) siRNAs to cisplatin-induced apoptosis was assessed, as described in figure 2D. The results of similar experiments presented in figure 2D for HCT116 cells (open symbols, dashed lines) are provided for direct comparison. (E-G) The activity of caspases 3, 8 and 9, was determined following exposure to 10 μM cisplatin. Each value in B-G represents the mean (± SEM) determined from a minimum of 3 independent experiments. An * in C indicates that the value is significantly less than 100% (P ≤ 0.05, single sample t-test) while an * in D indicates that the value is significantly different from its respective NT control (P ≤ 0.05, t-test). A significant difference in caspase 3, 8 and 9 activity was detected among transfectants following cisplatin treatment (P = 0.0001, 0.04 and 0.002, respectively, ANOVA). Similar results were obtained following exposure to UV light (data not shown).