Figure 4: Loading of RAD51 onto RPA-protected DNA is significantly reduced.

(a) HeLa cells were infected with 10 IU of adenovirus 48 hr prior to receiving 10 Gy and collection at 3, 6, and 12 hr post irradiation. Cells were subjected to immunofluorescence for BRCA1 (red) and RAD51 (far red, pseudo-colored green). Shown are representative images from untreated, 3 and 6hrs post irradiation. (b) Quantification of the co-localized foci from (A). Cells with ≥ 3 overlapping BRCA1 and RAD51 foci were counted as positive, and >100 cells were counted in three biological replicate experiments. (student’s t-test, n = 3, mean ± SEM, see also Supplementary Fig. S3). (c) HeLa cells were treated, collected, stained for RPA2 and RAD51 and quantified as in (A) and (B). (student’s t-test, n = 4, mean ± SEM, see also Supplementary Fig. S4). (d) To examine time points beyond 12 hrs, MEF cells were treated with 10 Gy, harvested at 3, 6, 12, 18, and 24 hr post treatment, and subjected to immunofluorescence for RPA2 (red) and RAD51 (far red, pseudo-colored green). Shown are representative images from untreated and 6 hr after irradiation. (e) Quantification of the co-localized foci from (D) performed as in (C).