Figure 5

Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation. (A) 293T/f-hMSH5 and 293T/f-hMSH5^Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. (B) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa, molecular weight (Mr) in thousands. (C) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5^Y742F, and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. (D) Immunoblotting analysis of the effectiveness of hRad51 or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa, molecular weight (Mr) in thousands. (E) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t-test).