Figure 2

Resveratrol treatment of MEFs transiently stabilises H2AX, leading to reduced numbers of γH2AX foci. (a) H2AX expression in senescent MEFs (passage 8) treated with different concentrations of resveratrol. The dose-dependent effects of resveratrol were assessed 1 hr after treatment. (b) The time-dependent effects of 5 μM resveratrol on H2AX expression in senescent MEFs (passage 8). (c) H2AX expression in senescent MEFs (passage 8) treated with resveratrol (5 μM) and then fractionated into the chromatin and non-chromatin fractions before and 1.5 and 24 hr after treatment. Histone H3 (H3) and PCNA were used as controls. (d) Immunofluorescence analyses examining the effect of resveratrol (2.5 μM) on the number of γH2AX foci in senescent MEFs (n numbers are indicated in the graph). The dose-dependent effects of resveratrol were assessed 3 and 24 hr after treatment. (e) The effects of multiple resveratrol treatments (2 μM) on the number of γH2AX foci in senescent MEFs. Reductions in the number of γH2AX foci were assessed 24 hr (single treatment) and 72 hr (three treatments) after resveratrol exposure. Data are represented as the mean ± s.d. (n = 3 biologically independent experiments). (f,g) The effects of resveratrol (2.5 μM) on the numbers of merged γH2AX/53BP1 (f) and γH2AX/p-RPA (g) foci in senescent MEFs. The MEFs were treated as shown in the workflow (f). γH2AX, 53BP1 and p-RPA were detected by immunofluorescence (n numbers are indicated in the graph). The percentages of the γH2AX foci that merged with 53BP1 or p-RPA foci (mean ± s.e.) are indicated in each image. Data in the graphs are represented as the mean ± s.d. Scale bars, 10 μm. P-values were calculated by two-tailed Welch’s t-tests.