Fig. 2
Oxidative stress and MMP defects in OPA1 R445H iPSC-RGCs. (a) DHE fluorescence signal normalised to cell number (Hoechst fluorescence). Isogenic/R445H, n = 18 wells sampled from three independent differentiations. **** = p < 0.0001, Mann-Whitney test. Scatter plot shows the mean values +/- SD. (b) MitoSOX fluorescence signal normalised to cell number (Hoechst fluorescence). Isogenic/R445H, n = 18 wells sampled from three independent differentiations. *** = p < 0.001, unpaired t-test. Scatter plot shows the mean values +/- SD. (c) Quantification of SOD1 Western blots. Isogenic/R445H, n = 12 sampled from three independent differentiations. No significant differences were observed between genotypes, p = 0.671, Mann-Whitney test. Scatter plot shows the mean values +/- SD. Western blot data is available in Supplementary Fig. 1. (d) Representative live cell confocal images of MMP in TMRE-stained isogenic control and R445H iPSC-RGCs at baseline and after FCCP treatment. Scale bar, 10 μm. (e) Quantification of MMP expressed as the fold-change in TMRE fluorescence relative to isogenic control samples. FCCP was used as a negative staining control to subtract non-mitochondrial TMRE fluorescence. Isogenic, n = 11; R445H, n = 10 images sampled from three independent differentiations. **** = p < 0.0001, unpaired t-test. Scatter plot shows the mean values +/- SD. (f) Representative live cell confocal images of TMRE-stained isogenic control and R445H iPSC-RGCs at baseline, 10 and 20 min after addition of 1.5 µM oligomycin, and following FCCP treatment. Scale bar, 10 μm. (g & h) Quantification of fold-change in TMRE fluorescence over time in isogenic control and R445H neurons. Fluorescence intensity was normalised to baseline values (F0). 1.5 µM oligomycin and 1 µM FCCP were added at the indicated time points. FCCP was used as a negative staining control to subtract non-mitochondrial TMRE fluorescence. Grey lines show individual cells TMRE fluorescence intensity from a representative experiment, blue/red lines the mean average normalised TMRE fluorescence intensity. (i) Quantification of endpoint TMRE fluorescence in isogenic control and R445H iPSC-RGCs prior to FCCP addition. Isogenic/R445H, n = 4 images acquired from three independent differentiations. ** = p < 0.01, unpaired t-test. Scatter plot shows the mean values +/- SD