Fig 2: Mitochondrial transcription factor A (TFAM) knockdown enhanced mycoplasma infection in hepatocellular carcinoma (HCC) cells. (A, B) Representative confocal microscopy images of immunofluorescence staining with anti‐TFAM, anti‐dsDNA Abs, and DAPI in HCC cells with stable TFAM knockdown as indicated. Blue, DAPI; green, TFAM, wheat germ agglutinin (WGA), or p37; red, dsDNA. shCtrl, control shRNA; shTFAM, shRNA against TFAM. Scale bar, 40 μm. (C, D) Representative confocal microscopy images of immunofluorescence staining with WGA (a cell membrane dye), anti‐dsDNA Ab, and DAPI in HCC cells with stable TFAM knockdown. (E, F) Representative confocal microscopy images of immunofluorescence staining with anti‐p37, anti‐dsDNA Abs, and DAPI in HCC cells with stable TFAM knockdown

Fig 3: Mitochondrial respiration and mass assessment in MERRF fibroblasts treated with rapamycin. Wt-Fibroblasts, I-Fibroblasts and H-Fibroblasts were treated four weeks with 20 nM rapamycin. A Representative Western Blot of p-S6, S6 and LC3 proteins. ACTIN was used ad loading control. B Densitometric analyses of downstream target of mTORC1, p-S6 protein. C Densitometric analyses of LC3-II protein. D OCR expressed as pmoles O2/min normalized to cell number, under basal conditions and after injection of oligomycin (O), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; F), rotenone (R) and antimycin A (AA). E Basal, ATP-linked and maximal respiration were calculated from OCR traces and reported in the graph. Data are means ± SEM of five experiments for Wt- and H-fibroblasts and of three experiments for I-fibroblasts. F Western Blot analyses of OXPHOS proteins, VDAC and TFAM mitochondrial mass proteins. One representative experiment is shown. Densitometric analyses of OXPHOS G and VDAC and TFAM proteins H. I mtDNA content evaluation by qPCR. J m.8344A > G mutation heteroplasmy evaluation by SNaPshot method. All data are normalized to untreated cells and, if not specifically indicated, are means and SD of six biological replicates for Wt-F, two biological replicates for I-F and four biological replicates for H-F. Statistical analyses were performed using unpaired two-tail T-test. P value: *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Transiently PGC-1α overexpression in MERRF fibroblasts. Wt-fibroblasts and H-fibroblasts were transiently transduced (72 h) with lentiviral vectors to generate either empty or over-expressing PGC-1α fibroblasts lines. A PGC-1α gene expression was evaluated by qPCR. ACTIN was used as reference gene. B Western blot of PGC-1α; ACTIN was used as loading control. A representative blot of three independent experiments (biological replicates) is shown. C mtDNA content evaluation by qPCR. D m.8344A > G mutation heteroplasmy evaluation by SNaPshot method. E ND1, F COX2 and G ATP6 gene expressions were evaluated by qPCR. ACTIN was used as reference gene. Data are means and SEM of three biological replicates and are normalized to the cells transduced with the empty plasmid. H Western Blot of TOM20, VDAC e TFAM mitochondrial mass proteins. ACTIN was used ad loading control. One representative of three independent experiment (biological replicates) is shown. I Densitometric analysis of TOM20, VDAC e TFAM mitochondrial mass protein. J Western Blot of OXPHOS proteins. ACTIN was used ad loading control. One representative of three independent experiment (biological replicates) is shown. K Densitometric analysis of OXPHOS subunits protein levels. L OCR expressed as pmoles O2/min normalized for protein content, under basal conditions and after injection of oligomycin (O), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; F), rotenone (R) and antimycin A (AA). M Basal, ATP-linked and maximal respiration were calculated from OCR traces and reported in the graph. All data are means and SEM of three independent experiments, analyzing three biological replicates, and are normalized to cells transduced with the empty plasmid. Statistical analyses were performed using unpaired two-tail T-test. P value: * p < 0.05, ** p < 0.01, *** p < 0.001

Fig 5: Abundance of mtDNA‐associated TFAM and TFB2M in patient and control (Cnt) substantia nigra neurons. (A) Epifluorescence microscopy after triple‐label immunofluorescence indicated joint loss of NDUFB8 and TFAM in idiopathic Parkinson disease (IPD) patients, whereas porin staining showed normal mitochondrial mass. (B) Densitometric analysis of investigated proteins confirmed significantly reduced TFAM:porin ratios in IPD patients. Lines represent mean group ratios. (C) In patients and controls, NDUFB8 and TFAM strongly correlated at the single‐neuron level, resulting in similar behaviors of the frequency histograms for NDUFB8:TFAM. (D) Triple‐label immunohistochemistry showed reduced abundances of NDUFB8 and TFB2M but normal porin levels in IPD neurons. (E) Densitometric analysis of average neuronal TFB2M and porin levels in patients and controls revealed a significant reduction of TFB2M:porin in the former. (F) The frequency distribution of the NDUFB8:TFB2M ratio in single neurons showed a slight shift toward lower TFB2M in patient cells. *p < 0.05. NDUFB8 = NADH dehydrogenase 1 beta subcomplex 8; TFAM = mitochondrial transcription factor A; TFB2M = mitochondrial transcription factor B2.