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Fig. 5 | Molecular Medicine

Fig. 5

From: Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys

Fig. 5

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

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