Fig. 1

Rett syndrome primary fibroblasts recapitulate alterations in mitochondrial homeostasis. We have focused the analysis on mitochondrial shape (A–H) and energy production (I–N). Mitochondrial network shape was studied by immunofluorescence of mitochondrial marker TOMM20 and parametrization with “Mitochondria Analyzer” plugin; A representative image of mitochondrial network IF (TOMM20 in yellow, nuclei in blue, and tubulin in grey; scale bar 10 µm; 6× zoomed section included in the white box) and (B) parametrization of number of independent networks corrected by area and mean form factor (a shape measure given by: P^2/(4piA): 1 indicates round object and increases with elongation, expressed as mean FF of objects in image), number of branches per mitochondrial network, and mean branch length and diameter. C–E Analysis and densitometry quantification of mitochondrial dynamics associated proteins Mfn2, OPA1, Drp1 and Fis1 by Western blot and corrected by vinculin. F Quantification of the fission and fusion events from in vivo imaging of mitochondrial network. Analysis of mitochondrial ultrastructure was performed by transmission electron microscopy; G representative images (scale bar 300 nm) and H parametrization of number of cristae per mitochondria, cristae length and width, and mitochondrial major:minor axis ratio. I Non-glycolytic ATP production –assumed as mitochondrial ATP– measured by luciferin-luciferase luminescence upon incubation with 2-deoxyglucose. J Mitochondrial respiration profile was measured by Seahorse in the presence of oligomycin, FCCP and Antimycin A. Oligomycin Sensitive Respiration (OSR) was calculated as the difference of oxygen consumption before and after adding oligomycin (considered as ATP-linked respiration). K O₂^.− production measured by flow cytometry with the MitoSOX probe L Expression of antioxidant enzymes MnSOD and GPX by Western blot, and M quantification of densitometry corrected by vinculin. N Oxidative damage was detected based on the fluorescence of RNA oxidative damage marker 8-OHdG, showed with the intensity-revealing LUT “royal” (calibration bar shown at the lower right corner of the panel), at 63× magnification; scale bar represents 10 µm, and represented as relative frequency of intensity values, calculated with Fiji software. All experiments were done in at least two different Rett and control cell lines, with three technical replicates and each experiment was done in triplicates. Statistical analyses were performed as described in Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Absence of asterisk means no statistically significant difference. Control fibroblasts in grey; Rett patients’ fibroblasts in yellow