Figure 5.

ACO2-mutant fibroblasts are more susceptible to oxidative stress. (a) Representative images of control and ACO2-mutant fibroblasts stained with MitoTracker green FM for live cell imaging of mitochondria. Images were obtained with a 40 × objective. Scale bars indicate 50 µm. Images were used to analyse parameters of mitochondrial morphology, indicating (b) mitochondrial length, reflected by aspect ratio and (c) mitochondrial branching, reflected by form factor. Data indicated as mean ± SEM (n = 4). (d) Mitochondrial membrane potential of fibroblasts was analyzed by FACS. Cells were treated with 5 nM Valinomycin for 14 h in order to decrease the mitochondrial membrane potential and afterwards stained with TMRE. Data indicated as mean ± SEM, significance calculated by 2way ANOVA with post hoc Tukey’s multiple comparison test (n = 4). (e) Mitochondrial superoxide production was measured in fibroblasts using FACS analysis of MitoSOX staining. Cells were first treated with 20 nM Piericidin A for 14 h in order to inhibit the activity of the respiratory chain complex I. Data indicated as mean ± SEM, significance calculated by Mann–Whitney test (n = 3). (f) Cell viability was assessed using the Lactic Acid Dehydrogenase (LDH) assay. Fibroblasts were incubated with 5 mM H₂O₂ for 4 h and afterwards the proportion of cell death was calculated from the amount of released LDH (n = 5). Data indicated as mean ± min./max. values. Significance calculated by Mann–Whitney test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.