Fig. 1
From: Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation
Loss of RAD52 causes MRE11-dependent accumulation of nascent ssDNA. a Western blot shows level of RAD52 protein in cells were infected with two different viruses (V1 or V2) containing short hairpin RNA (shRNA) sequences against RAD52. LAMIN B1 was used as loading control. C = control virus. b Analysis of nascent ssDNA after 2 mM hydroxyurea (HU) treatment. On top: schematic of the experiment. Graph shows the intensity of ssDNA staining for single nuclei. Values are presented as means ± SE (ns = not significant; ****P < 0.0001; Mann–Whitney test; N = 184). Representative images are shown. c Western blot analysis of FLAG-RAD52 transfection. LAMIN B1 was used as a loading control. d Cells were transfected with FLAG-RAD52 or FLAG-empty 48 h before to perform nascent ssDNA immunostaining. Dot plots show the mean intensity of ssDNA staining for single nuclei from each cell line after treatment with HU 2 mM. Values are presented as means ± SE (****P < 0.0001; Mann–Whitney test; N = 184). Representative images are shown. e Cells were treated with 2 mM HU at different time points and collected to perform neutral comet assay. Data are presented as mean tail moment ± SE from two independent experiments. N = 93 (ns = not significant; *P < 0.1; ****P < 0.0001; Mann–Whitney test). Representative images are shown. f Cells were treated as indicated in the schemes. Graph shows the mean intensity of ssDNA staining for single nuclei from each cell line. The intensity of the anti-iododeoxyuridine (IdU) immunofluorescence was measured in at least 100 nuclei from two independent experiments. Values are presented as means ± SE (ns = not significant; *P < 0.1; ****P < 0.0001; Mann–Whitney test; N = 290). Representative images of ssDNA formation are given. Scale bar represents 5 µm. Source data are provided as a Source Data file
