Fig. 5

Rad52 increased expression, recruitment, and foci formation at DSBs upon prolonged p21^WAF1/Cip1 expression. a Immunofluorescent (IF) analysis showing increased Rad52 foci formation in 96-h induced Saos2- and Li-Fraumeni-p21^WAF1/Cip1 Tet-ON cells. Immunoblot (IB) and real-time RT-PCR assessment of Rad 52 expression levels in induced Saos2- and Li-Fraumeni-p21^WAF1/Cip1 Tet-ON cells at the indicated time point (*p < 0.05 (Saos2), *p = 0.05 (Li-Fraumeni), t-test; error bars indicate standard deviation; n = 3 experiments). b IF analysis showing Rad52 and RPA foci formation and their co-localization in 96-h induced Saos2- and Li-Fraumeni-p21^WAF1/Cip1 Tet-ON cells. Saos2- and Li-Fraumeni-p21^WAF1/Cip1 Tet-ON cells were pre-extracted with ice-cold PBS containing 0.2% Triton X-100 for 2 min on ice before fixation as previously described [52]. c Timelapse microscopy showing Rad52 loading on regions of damaged chromatin after UV-laser ablation in 96-h induced Saos2- and Li-Fraumeni p21^WAF1/Cip1 Tet-ON cells transfected with the YFP-Rad52 vector. Plots depict Rad52 recruitment kinetics at sites of DNA damage in the same cells, respectively. The average intensity of fluorescence at the site of damage and the total cell fluorescence in respect to time were quantified and plotted. Five cells in each condition of two independent experiments were processed. d Rad52 promoter is occupied by E2F1 upon p21^WAF1/Cip1 induction in Saos2- and Li-Fraumeni-p21^WAF1/Cip1 Tet-ON cells, respectively, as assessed by chromatin immunoprecipitation (ChIP; *p < 0.05, t-test; error bars indicate standard deviation; n = 3 experiments; see also Additional file 1: Figure S8). e Silencing of E2F1 resulted in decreased Rad52 levels as assessed by immunoblot analysis in induced Saos2- and Li-Fraumeni-p21^WAF1/Cip1 Tet-ON cells, respectively (*p < 0.01, t-test; error bars indicate standard deviation; n = 3 experiments). Actin serves as loading control; (Ctl) siRNA; arrows indicate Rad52