Fig. 3
From: Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation
Accumulation of nascent ssDNA in RAD52i cells is dependent on fork reversal. a Cells were treated as indicated in the scheme. Graph shows the mean intensity of ssDNA staining at least 100 nuclei. Values are presented as means ± SE (ns = not significant; ****P < 0.0001; Mann–Whitney test; N = 475). b Analysis of RuvA recruitment in chromatin after replication stress. H3 was used as a loading control. c Cells were infected with tetracycline-inducible virus (V1) containing short hairpin RNA (shRNA) sequences direct against SMARCAL1 to produce MRC5 shSMARCAL1 inducible cells lines. Cells were treated or not with doxycicline for 40 h then was labelled with iododeoxyuridine (IdU) to detect nascent ssDNA in presence or not of RAD52 inhibitor and or 2 mM hydroxyurea (HU) for 4 h. Graph shows the mean intensity of ssDNA staining at least 100 nuclei. Values are presented as means ± SE (**P < 0.1; ***P < 0.001; Mann–Whitney test; N = 135). d Cells were transfected with scrambled siRNA (siCTRL) or siRNA directed against ZRANB3. Western blot analysis shows level of protein. LAMIN B1 was used as a loading control. e Graph shows the main intensity of ssDNA staining for single nuclei from untreated or treated cells. Values are presented as means ± SE (**P < 0.1; ****P < 0.0001; Mann–Whitney test; N = 142). Representative images are given. f Analysis of nascent ssDNA in cells inhibited or not for RAD51 and/or RAD52. Graph shows the main intensity of ssDNA staining for single nuclei from untreated or treated cells. Values are presented as means ± SE (****P < 0.0001; Mann–Whitney test; N = 278). g Experimental scheme of nascent ssDNA. Analysis of IdU intensity in cells treated or with RAD51 in combination or not with RAD52 inhibitor. When indicated the inhibitors were added at different time point. Dot plots show the mean intensity of ssDNA staining for single nuclei from each cell line after treatment with HU for 4 h. Mean values are represented as horizontal black lines ± SE; N = 164. (ns = not significant; **P < 0.1; ***P<0.001; ****P < 0.0001; Mann–Whitney test; N = 475). Scale bar represents 5 µm. Source data are provided as a Source Data file
