Figure 3(A) Evaluation of ssDNA accumulation at parental-strand by immunofluorescence microscopy analysis in wild-type (shWRNIP1WT) or WRNIP1-deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity is chemically inhibited with 50 µM Mirin, alone or in combination with HU-induced replication stress. After treatment, cells were fixed and stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines represent the mean SE; n = 3. Error bars represent standard error (ns, not significant; **, p<0.01; ****, p<0.0001; two-tailed Student's t test). Representative images are shown. DNA was counterstained with DAPI (blue).(B) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti-WRNIP1, anti-MRE11 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (Input) in the cells was determined with the relevant antibodies. LAMIN B1 was used as a loading control. In the graph, the fold increase respect to the wild-type untreated of the normalized ratio of the chromatin-bound RAD51 (or MRE11)/ total RAD51 (or MRE11) is reported for each cell line.(C) Analysis of DNA-protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay. Experimental designed used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental-strand ssDNA, and subjected to PLA assay as described in the "Experimental procedures" section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively. Each red spot represents a single interaction between ssDNA 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 given. Graph shows the number of PLA spots per cell. Horizontal black lines represent the mean value (ns, not significant; **, p < 0.01; two-tailed Student's t test); n = 3.(D) Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU-co-immunoprecipitation (CldU-IP). shWRNIP1WT or shWRNIP1 cells were pulse-labelled with CldU, then fixed or treated with HU. Cells were cross-linked, and the nuclear extracts were isolated (Input) and subjected to CldU-IP using an anti-CldU antibody (CldU-IP). The membranes were probed with the anti-WRNIP1 or anti-RAD51 antibodies. After stripping, the membranes were probed with an anti-MRE11 antibody. LAMIN B1 and GAPDH were used as loading controls (Input). Ponceau S was used as a loading control of CldU-IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti-CldU antibody. The graph shows the normalized ratio of the proteins co-immunoprecipitated with CldU (CldUCo-IP proteins)/ the total of labelled DNAimmunoprecipitated with CldU (CldUIP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2.