Figure 6

Targeting HRR is more efficient than targeting NER to sensitize cisplatin-resistant ovarian cancer cells to cisplatin. A) PEO4 cells were transfected with control siRNA, XPA siRNA, BRCA2 siRNA separately, or both XPA and BRCA2 siRNA for 48 h. Whole cell lysates were prepared and subjected to Western blotting for detecting the expression of XPA and BRCA2. B) PEO4 cells were transfected with various siRNA as described in (A) for 24 h, pretreated with HU for 24 h, and then treated with cisplatin for 1 h. The cells were then incubated in HU containing medium for 0, 8 and 24 h. The genomic DNA was isolated and analyzed by ISB assay for cisplatin-induced 1,2-intrastrand crosslinks with anti-Pt-GG antibody. The relative percentage of remaining Pt-GG at different time points is an average of three independent repeats (n = 3, Bar: SD). C) PEO4 cells growing on coverslips were transfected with various siRNA as described in (A) for 48 h, irradiated with X-ray at 10 Gy, and further cultured for 1 and 24 h. the γH2AX-positive cells were detected with immunofluorescence as described in Materials and Methods. D) PEO4 cells growing in 96-well plates were transfected with various siRNA as described in (A) for 24 h. Cells were then treated with cisplatin at the indicated dose for 1 h, and further cultured in drug-free medium for 72 h. Methylene blue stained cells in the treated samples compared to untreated control cells were used to obtain relative cell survival. (n = 4, Bar: SD).