WRN -mutated fibroblasts showed nuclear shape abnormalities, oxidative stress and premature senescence, without altered expression and localization of type A lamins. Cultured skin fibroblasts from patient 1 were compared to control cells. (A) Nuclear staining of fibroblasts was performed with diamidino-2phenylindole hydrochloride (DAPI) (DNA, blue), and with antibodies directed against lamin A/C (red), and lamin B (green). Representative cells, studied at passages 4 to 8, are shown. Lamin A/C staining was heterogeneous and lamin B staining reduced or absent in nuclear blebs or poles of dysmorphic WRN-mutated cells, but not in control cells. Scale bars represent 10 μm. Quantification of cells with dysmorphic nuclei was performed after examination of 200 to 250 cells for each subject. Results are expressed as mean ± SEM. ***, p < 0.001. (B) Fibroblast lysates were submitted to Western blot, using antibodies which specifically detect lamin A/C (MAB3211), lamin B1 (ab16048), prelamin A (SC-6214), and uncleaved prelamin A (having retained its CSIM C-terminal tail) (ANT0045). Beta-actin was used as a loading control. Representative results from control, WRN-mutated, and p.R482W LMNA-mutated cells, used as positive controls , are shown as indicated. (C) ROS production was assessed by oxidation of CM-H2DCFDA derivatives, normalized to the DNA content, and senescence-associated ß-galactosidase cell activity by the ratio of X-gal staining at pH6 and pH4, in WRN-mutated and control fibroblasts studied at the same passage (2 to 5). Results from four experiments performed in quadriplate are shown, as mean ± SEM. *, p < 0.05, ***, p < 0.001. (D) Representative micrographs of cells stained with X-gal at pH 6 are shown. Control cells were studied at passage 1 to 4, and WRN-mutated cells at passage 1. Note the senescence-associated blue staining, flattened and enlarged morphology of WRN-mutated cells. Scale bars represent 40 μm.