Fig 2: Characterization of Mfn1-ßIIPKC protein–protein interaction. Recombinant Mfn1 (GST-Mfn1, 25 ng) was incubated with recombinant ßIIPKC (GST-ßIIPKC, 25 ng) in the presence and absence of phosphatidylserine and diacylglycerol (PS/DAG/Ca2+, PKC activators) for 30 min at 37 °C. a Co-immunoprecipitates with anti-Mfn1 and ßIIPKC were analyzed by western blot. b Mfn1 phosphorylation was evaluated using radioactive [32P] 32P-ATP incorporation after incubation with either ßIIPKC or its alternative splicing ßIPKC in the presence and absence of classic PKC activators. Representative blots of three independent experiments. c GTPase activity of Mfn1 determined in the presence of active ßIIPKC or its alternative splicing ßIPKC (n = 6 per group). d Conservation of the ßIIPKC phosphorylation in human orthologs of Mfn1 in rat, mouse, and zebrafish and Mfn2 in mouse. Ribbon presentation of the 3D structure of Mfn1 phosphorylation at the ßIIPKC site (PDB: 5GOM). e Individual replacement of serine by alanine at residues 86, 284, and 290 in Mfn1 knockout MEFs. Cells were transfected with the following constructs: WT, S86A, S284A and S290A. 48 h after transfection cells were incubated with H2O2 (100 µM, 24 h) and measured cytotoxicity (LDH release, n = 6 per group). Data are means ± SEM. *P < 0.05 vs. control. One-way analysis of variance (ANOVA) with post-hoc testing by Duncan

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-1a 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

Fig 4: DNMT1 localization in mitochondria and mtDNA methylation analysis. (A and B) Western blot of DNMT1 localization in total lysate (TL), nuclear (NF), cytoplasmic (CF) and mitochondrial (MF) fractions of primary or immortalized fibroblasts (A) or HeLa and SK-N-SH cells (B). HDAC1 and COXIV were used as markers for nuclear or mitochondrial fractions, respectively. IMM: immortalized fibroblasts. (C) Protease K protection assay on purified mitochondria in HeLa and SK-N-SH. MFN2, TIM23 and CS were used as a marker of outer membrane, inner membrane and matrix. PK: proteinase K (ug); DIG: digitonin. (D and E) Mitochondrial D-loop CpG methylation, assessed by bisulfite-NGS, after linearization of mtDNA with BamHI digestion. Data are expressed as means ± SEM of % of methylated cytosines on total mtDNA molecules sequenced, for each nucleotide position of L-strand (D) and H-strand (E).

Fig 5: Levels of mitochondrial shaping proteins in MEFs bearing the indicated OPA1 mutations incubated with ORMs. MEFs were incubated for 24 h in DMEM-galactose in the absence or presence of ORMs. (A, D, F, H) Representative blots of OPA1, MFN2, DRP1 and DRP1-P-S616. ACTIN or TUBULIN were used as a loading control. (B, E, G) Densitometric analysis of OPA1, MFN2 AND DRP1 protein levels. Data are normalized to those of untreated cells in DMEM-galactose. Values are means ± SEM (n = 3–6). *P < 0.05, one simple t test. (C) Densitometric analysis of OPA1 long-/short-forms ratio. Values are means ± SEM (n = 4–6). (I) Densitometric analysis of DRP1-P-S616/DRP1 ratio. Values are means ± SEM (n = 3).