Figure 6(A) Analysis of DNA damage accumulation. Wild-type (shWRNIP1WT), WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells were treated or not with 4 mM HU for 4 h, then subjected to -H2AXimmunofluorescence microscopy. Graph shows data presented as mean of -H2AX-positive cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of nuclei showing the different number of foci per nucleus are reported.(B) Analysis of DNA breakage accumulation. shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells were treated as in (A), then subjected to alkaline Comet assay. Graph shows data presented as mean tail moment SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images are shown.(C) Evaluation of cell death. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h. Cell viability was evaluated by LIVE/DEAD fluorescent assay. Data are expressed as mean of dead cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of double-staining of viable (green) and dead (red) cells are shown.(D) Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU, then left to recover for 16h in drug-free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.(E) Experimental scheme of the Mirin aberration analysis is given. The experiment was carried out as in (D) but cells were pre-treated or not with 50 µM Mirin. Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.(F) Experimental design of the chromosomal aberration assay is reported. shWRNIP1 cells were transfected with control siRNAs (siCtrl) or FBH1 siRNA (siFBH1). Fourth-eight hours thereafter, cells were treated or not with 4 mM HU and then left to recover for 16 h. Metaphases were collected with colcemid and prepared as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Horizontal black lines represent the mean SE. Error bars represent standard error. (**, p < 0.01; two-tailed Student's t test).