Figure 3.

Altered mitochondrial function and increased oxidative stress in WDR45 mutant fibroblasts. (A) The mitochondrial network was investigated under basal conditions by fluorescence microscopy in fixed cells immunostained with anti-GRP75 (green). The form factor as a measure for mitochondrial interconnectivity was calculated for two control (ctrl) fibroblast lines (n = 72) and one patient line (n = 47). The median, the interquartile range, and the minimum and the maximum value of the investigated individuals are shown. (B) Mitochondrial membrane potential was analysed by using a fluorescent dye and normalized to protein concentration (n = 4 independent experiments). (C) ATP production was determined based on the mitochondrial oxygen consumption rate using an extracellular flux analyser (n = 4 independent experiments). (D) Western blot analysis was performed on samples under basal conditions and upon treatment with 1-methyl-4-phenylpyridinium (MPP+) or ferric chloride (FeCl₃). The western blot was probed with antibodies against superoxide dismutase 2 (SOD2) and β-actin (loading control). Statistical significance was analysed by selected unpaired t-tests. (E) Oxidation of proteins in WDR45 mutant fibroblasts and controls was determined by OxyBlot using an antibody against DNP. Statistical significance was analysed by selected unpaired t-tests. (F) Western blot analysis for mitochondrial ferritin (FtMt) precursor protein at 30 kDa. The antibody did not detect the processed protein at 22 kDa (n = 2 independent experiments).