Methylene blue restores H3K9me3 and rescues DDR defects in HGPS.

<p>(A). Western blotting analysis with anti-pATM(S1981), anti-ATM, anti- gammaH2AX, anti-HP1alpha, anti-H3K9me3 and anti-GAPDH antibodies on late passage normal and HGPS fibroblasts treated with or without MB after 30 days. Cells were treated with Dox to induce DSBs before analysis. Two independent western blotting analysis were performed. (B). Representative fluorescence images of gammaH2AX foci and 53BP1 foci in normal and HGPS fibroblasts treated with or without MB for 30 days. Cells were treated with Dox to induce DSBs before analysis. Scale Bar: 5um. (C). Line profile analysis of (B), showing the robust recruitment of 53BP1 to gammaH2AX in MB treated HGPS fibroblasts. Green (gammaH2AX) and red (53BP1) fluorescence intensities (y axis) were plotted against distance (x axis) along the yellow line in (B). Stars indicated strong co-localization of 53BP1 to gammaH2AX. (D). Representative fluorescence images of gammaH2AX foci in late passage normal and HGPS fibroblasts treated with or without MB after 30 days. Cells were then treated with Dox to induce DSBs and allowed to recover for indicated amounts of time before analysis. Scale Bar: 5um. (E). Quantification of (D), showing the number of gammaH2AX foci in each nucleus at each time point. More than 100 cells were randomly picked for quantification in each group. Results were presented as mean ± SEM. ***P < 0.001.