Fig. 5: MTX2 deficiency induces similar nuclear defects in patients’ fibroblasts and C. elegans cells.
a Proliferation rates were analyzed by the determination of doubling times in hours (n = 12 independent experiments). Results are expressed as mean ± SD. Statistical significance was analyzed using two-tailed Mann–Whitney test (95% CI); ***p < 0.001 (exact p values: pMADM2 = 7.39E−07; pMADM3 = 1.47E−06). b Senescence levels were assessed by SA-β-galactosidase quantification on n = 225 (WT), 183 (MADM2), 168 (MADM3) and 171 (HGPS) cells at the same passage number from one experiment. Scale bar, 25 µm. c Fluorescence images of nuclei labeled by DAPI staining, showing nuclear morphological abnormalities. Scale bar, 50 µm. d Quantification of cells with abnormal nuclear morphology. The average percentage mean values of normal and abnormal nuclei was calculated as described; n = 150 independent cells for each cell line at the same passage number were counted from n = 3 independent experiment. Chi-square test was used (exact p values: pMADM2 = 3.16E−13, pMADM3 = 1E−15, pHGPS = 1E−15); ***p < 0.001. e A representative western blot of fibroblasts’ whole lysates showing Lamin A/C, progerin vs. actin expression in control (WT), MADM2, MADM3, and HGPS from one experiment. f Immunofluorescence staining of Lamin A/C (green), Mitotracker (red), and DAPI (blue) in patients’ and control fibroblasts showing nuclear blebbing and aberrant folding with altered Lamin A/C staining. The images are representative of n = 3 independent experiments. Scale bar, 10 µm. g mtx-2 downregulation by siRNA causes nuclear aggregates in C. elegans. Wild-type C. elegans expressing lmn-1:gfp (Ce-lamin-GFP) were transfected with either mtx-2 RNAi or empty vectors (EV). Representative images of Ce-lamin-GFP nuclear aggregates monitored at days 2, 5, and 7 after transfection. Scale bar, 10 µm. h Graphical representation of the number of nuclear aggregates in aging animals transfected with either empty vector (EV) or mtx-2 siRNA (mtx-2); Y axis: percent of nuclei in each of three categories (class I: 0 aggregates, class II: 1–5 aggregates, class III: >5 aggregates), X axis: day of adulthood (2, 5, 7) upon transfection; (n = 145–148 nuclei were counted from ten independent animals for each condition; average: 14,6 nuclei per worm, cf. Source Data File). Fisher’s exact test p values: ***pday2 = 1.14E−04, ***pday5 = 1.00E−08, **pday7 = 0.00242572; *p < 0.05, **p < 0.01, ***p < 0.001). i Representative images of the different mitochondrial morphologies observed, subdivided in normal and abnormal as described. Lower panels: magnification of the region surrounded by a rectangle in the upper panels. Scale bar: 5 µm. j quantification of mitochondrial morphologies in mitogfp strain (n = 17 biologically independent animals) and mtx-2 KO; mitogfp strain (n = 68 biologically independent animals). The data are representative of two independent experiments. Source data are provided as a Source Data file.
