Figure 4

MB improves HGPS nuclear morphology and solubilizes progerin. (A) Growth curves of normal‐1 and HGPS‐1 fibroblasts treated with vehicle or methylene blue (MB) at 100 n m for 12 weeks. The treatment started at the passage 14 for all cell lines and ended at passage 20 when the HGPS‐1 cells in mock treatment reached senescence. (B) Representative immunofluorescence images of HGPS‐1 and HGPS‐2 fibroblasts treated with vehicle or MB 100 n m for 8 weeks. Red: antilamin A/C antibody, Green: antiprogerin antibody. Scale bar, 20 μm. (C) Quantification of mean negative curvature (MNC) in HGPS‐1 and HGPS‐2 fibroblasts treated with vehicle or MB at 100 n m for 8 weeks. Over 100 nuclei from each group were randomly selected, and their boundaries were quantitatively analyzed using the nuclear morphology analysis software that was reported previously (Driscoll et al., ). The number of nuclei in each group is indicated in the parentheses underneath each cell type (**P < 0.01). (D) Quantitative RT–PCR analysis of lamin A, progerin and lamin C mRNA levels in HGPS‐1 fibroblasts treated with vehicle or MB at 100 n m for 6 weeks (*P < 0.05). (E) Left: Western blotting analysis with antilamin A/C and anti‐β‐actin antibodies in HGPS‐1 fibroblasts treated with vehicle or MB 100 n m for 6 weeks. Two technical replicates per sample were shown. Right: Quantification of relative protein amounts of lamin A, progerin, and lamin C. (F) Nuclear fractionation and Western blotting showing the input, soluble and insoluble fractions of lamin A, progerin and lamin C. Normal‐1 and HGPS‐1 fibroblasts were in treatment with vehicle or 100 n m MB for 6 weeks. (G) Percentages of soluble (Green) and insoluble (Orange) fractions of lamin A (including prelamin A) and lamin C in normal‐1 cells. Data for each protein were collected based on the band intensities. Sol% = Sol/(Sol + Insol) X 100%; Insol = Insol/(Sol + Insol) X 100%. (H) Pie graphs showing the combined analyses of soluble and insoluble fractions of lamin A and lamin C (A+C) in normal‐1 cells. (I) Percentages of soluble (Green) and insoluble (Orange) fractions of lamin A (including prelamin A), progerin and lamin C in HGPS‐1 cells. Data for each protein were collected based on the band intensities. Sol% = Sol/(Sol + Insol) X 100%; Insol = Insol/(Sol + Insol) X 100%. (H) Pie charts showing the combined analyses of soluble and insoluble fractions of lamin A, progerin, and lamin C (A+P+C) in HGPS‐1 cells.