Figure 5(A) Co-immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG-WRNIP1 plasmid. Cells were treated or not with HU. After treatment, cell lysates were immunoprecipitated (FLAG-IP) using anti-FLAG antibody. The presence of WRNIP1, BRCA2 and RAD51 was assessed by immunoblotting using the anti-FLAG, anti-RAD51 and anti-BRCA2 antibodies, respectively. Whole cell extracts were analysed (Input). The membrane was probed with the same antibodies used for IP. GAPDH was used as a loading control. (B) Analysis of protein-protein interactions between WRNIP1 and endogenous RAD51 in wild-type (shWRNIP1WT) or WRNIP1-mutant (shWRNIP1T294A) cells by in situ PLA assay. Cells were labelled with IdU for 24 h, washed and left to recover for 2 h, then treated or not with 4 mM HU. Antibodies raised against FLAG-Tag and RAD51 were used to reveal FLAG-WRNIP1 or endogenous RAD51 respectively. Each red spot represents a single interaction between WRNIP1 and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are shown. Graph shows the mean number of PLA spots per cell SE. Error bars represent standard error (ns, not significant; two-tailed Student's t test); n = 3. (C) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were transfected with BRCA2 siRNA (siBRCA2), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1WT/siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells. The membrane was probed with an anti-BRCA2 or anti-WRNIP1. GAPDH was used as a loading control. (E) Experimental scheme of pulse-labelling of DNA fibers in shWRNIP1 cells. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(F) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. (G) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h treated or not with HU for 4h. Chromatin fractions of cells were analysed by immunoblotting. The membrane was probed with the anti-FBH1 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 (Input) in the cells was determined with the relevant antibodies. GAPDH was used as a loading control. The ratio of the RAD51/LAMIN B1 signal (chromatin) is reported below each lane.