Figure 5.

Depletion of MUTYH confers oxidative stress tolerance to WRN- and Polλ-deficient cells and impairs the recruitment of WRN and Polλ to sites of oxidative DNA damage. (A–D) Graphs showing sensitivity of siRNA-depleted HeLa cells to indicated concentrations of H₂O₂. siCtrl, siMUTYH, siWRN and siPolλ represent siRNA against luciferase, MUTYH, WRN and Polλ, respectively. Cell viability assays were performed as described in the ‘Materials and Methods’ section. Each data point represents mean ± SD (n = 12). (E) Western blot analysis of extracts of HeLa cells transfected with different siRNAs as indicated. W, P and M stand for WRN, Polλ and MUTYH, respectively. Cells were harvested 72 hours after transfection. Blots were probed with antibodies against MUTYH, WRN, Polλ and TFIIH (loading control). IB, immunoblotting. (F) Effect of MUTYH depletion on nuclear focus formation of WRN and Polλ in U2OS cells after oxidative stress. Cells were treated with 500 µM H₂O₂ for 2 h, fixed and immunostained as in Figure 3A. H₂O₂-treatment was carried out 72 h after siRNA transfection. The data points represent the mean of three independent experiments with at least 100 nuclei scored in each experiment. (Right panel) Western blot analysis of extracts of U2OS cells transfected with siCtrl and siMUTYH, respectively. Cells were harvested 72 h post-transfection. Blots were probed with antibodies against WRN, Polλ, MUTYH and TFIIH (loading control).