Fig 1: Resveratrol activates ATM in human primary fibroblasts (GM08399) in combination with H2O2 or bleomycin.(a, b) The effects of resveratrol on human primary fibroblasts were tested as in Figure 1 but with varying levels of H2O2 or bleomycin as shown. (c) To deplete ATM, the fibroblasts were transduced with lentivirus expressing shRNA directed against ATM shRNA plasmid. After selection with puromycin, cells were tested for ATM expression and ATM target phosphorylation in combination with KU-55933, resveratrol, H2O2, and bleomycin as in Fig. 1. (d) Human primary fibroblasts were treated with resveratrol, bleomycin, or both as in (a) and probed for ?-H2AX foci by immunofluorescence. Cell images (82, 83, 78, and 82 cells, respectively, were analyzed for foci using Image J software, and the average number of foci per cell were quantitated. Error bars show standard error and * indicates comparisons in which p<0.05. (e) Human primary fibroblasts were treated as in (d) and the percentage of cells containing >5 foci was quantitated. Cell images from 3 independent experiments with a total of 266, 264, 248, and 269 cells, respectively, were quantitated. (f) Human primary fibroblasts were treated with resveratrol (100 µM), hydrogen peroxide, or both as in (b) and were quantitated for ?-H2AX foci by immunofluorescence. 107, 110, 104, and 106 cells, respectively, were analyzed for foci using Image J software, and the average number of foci per cell was calculated. (g) Quantitation of total pan-nuclear ?-H2AX signal per nucleus in cells treated with resveratrol, H2O2, and bleomycin as in Fig. 1. The average nuclear signal in untreated cells was normalized to 1. (h) Representative immunofluorescence images with fibroblasts treated as in (a). (i) Representative comet assay images with fibroblasts treated as in (a). (j) Quantification of comet tail length from fibroblasts treated as in (a); 30 cells were measured for each condition.