Fig. 4

Chronic treatment with SAG improves mitochondrial function and activity of FXN-deficient astrocytes. In A–H HAs were transduced with either the LV-scrambled or LV-shRNA37 for 96 h, SAG was added daily at 1 μM for a total of 6 days. A Representative histogram and quantification of the flow cytometry analysis of mitochondrial superoxide production using the MitoSOX Red dye (n = 3). B Analysis of mitochondrial oxygen consumption rates determined in HAs by the Seahorse assay in the indicated experimental conditions. The graph represents the OCR over time before and after the addition of the different drugs that modulate mitochondrial respiration (n = 4). C Normalization of the results showed differences in ATP coupled respiration, maximal respiratory capacity, basal mitochondrial respiration and spare capacity in FXN-deficient HAs and FXN-deficient cells + SAG when compared to the LV-scrambled-transduced cells (n = 4). D Representative energy map of all experimental conditions depicting OCR on the y-axis and ECAR on the x-axis (n = 4). E Quantification of total ATP production rate, showing the proportion of ATP that comes from glycolysis and that coming from the mitochondrial electron transport chain (n = 4). F Representative images of HAs stained with BODIPY 493/503 showing lipid droplet content and density. Scale bars: 30 μm. G Representative flow cytometry histogram showing lipid droplet accumulation in LV-shRNA37-transduced cells (n = 5). H Representative immunoblot and quantification of PLIN2 levels in all experimental conditions. Equal amounts of protein were immunoblotted and probed with antibodies against PLIN2 and β-actin (as loading control) (n = 6). Data in all graphs were normalized to the untreated group (data not plotted) and represent mean values ± S.E.M. In E $ refers to p-values of Mito ATP, whereas # refers to p-values of Glyco ATP. In A, C, E, G, H, data were evaluated by one-way ANOVA followed by Tukey’s post hoc test