Fig. 4.

Prior ATR inhibition increases the number of DNA double-strand breaks induced by hydrogen peroxide. a U2OS cells were incubated with IdU for 24 h before the experiment. EdU of 10 μM was added 15′ before 1 h treatment with 5 μM AZD/DMSO. Cells were fixed and stained with anti-BrdU antibody to detect ssDNA followed by click-chemistry reaction to visualize EdU. Scale bar represents 10 μm. b Quantification of the previous experiment. For Cdc7i samples, 10 μM Cdc7i was added at the same time as EdU. Error bars represent standard deviation. c BJ-hTERT fibroblasts were treated with vehicle or 5 μM ATRi (AZD6738) for 45 min before the addition of 15 μM H₂O₂ and 10 μM EdU to label replicating cells. Cells were fixed 30 min after adding H₂O₂. 53BP1 was detected by indirect immunofluorescence; EdU was detected by click-chemistry reaction. Representative images are shown. Scale bar represents 10 μm. d As in c, but Cdc7 inhibitor was added 15 min before ATRi. Numbers of 53BP1 foci per cell were determined. Three experimental repeats were performed and showed similar results; quantification of one is shown. e Schematic representation of a model: sequence of administration of ATRi and DNA damaging agent affects the number of induced lesions