Fig. 3

SAG positively modifies mitochondrial morphology and dynamics of FXN-deficient astrocytes. In A–H, HAs were transduced with either the LV-scrambled or LV-shRNA37 for 96 h, adding SAG (1 μM) daily for 6 days. A Representative confocal images of HAs stained with MitoTracker Red before fixation and imaging. Insets are twofold enlargements of the boxed region. Scale bars: 30 μm (expanded fields) and 15 μm (insets). B Analysis of mitochondrial morphology and distribution of MitoTracker Red positive cells using the MiNA plugin (ImageJ). Each plotted value represents a single cell from three independent experiments. C Representative immunoblots and quantification of Drp1, pDrp1, OPA1, MFN1 and MFN2 levels across the indicated experimental conditions (n = 4). D Representative microscopy images of HAs transduced with LV- shRNA37 that were treated or not with SAG. Cells were incubated with MitoTracker Red, fixed and then stained with pDrp1 and with To-Pro-3 to stain the nuclei (blue). Scale bars: 50 μm. E Representative immunoblot (left) and quantification (right) of LC3-I and its lipidated form LC3-II in the different experimental conditions before and after treatment with BafA1 (n = 3). F Quantification of autophagic flux of LC3 calculated as the ratio of LC3-II between BafA1 and vehicle in each experimental condition (n = 3). G Representative immunoblots and quantifications of PINK1 and Parkin levels in the indicated conditions (n = 5). H Representative immunoblot and quantification of PGC-1α levels in the different experimental conditions (n = 3). Data in all graphs were normalized to the untreated group (data not plotted). Except from B where data represent mean ± S.E.M of individual cells across experiments, the rest of the plotted data represent mean values ± S.E.M. In B, C, E–H data were evaluated by one-way ANOVA followed by Tukey’s post hoc test