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Fig. 4 | Molecular Medicine

Fig. 4

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

Fig. 4

PGC-1α steady over-expression in MERRF cybrids. Wt-cybrids and H-cybrids were transduced with either an empty or a PGC-1α expressing lentiviral vector and then puromycin-selected to generate steady over-expressing PGC-1α cybrids 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 Densitometry of PGC-1α protein content, normalized to the cybrids transduced with the empty plasmid. D ND1, E COX2 and F ATP6 gene expression evaluated by qPCR. ACTIN was used as reference gene. Data are means and SEM of three biological replicate and are normalized to the cells transduced with the empty plasmid. G Western Blot of OXPHOS subunits and of TOM20, VDAC e TFAM protein. ACTIN was used ad loading control. One representative of three independent experiment (biological replicates) is shown. H Densitometric analysis of OXPHOS subunits protein level. Data, normalized to the cybrids transduced with the empty plasmid, are means and SEM of six independent experiments (biological replicates). I Densitometric analysis of TOM20, VDAC and TFAM mitochondrial mass proteins. Data are means and SEM of three independent experiments (biological replicates) and are normalized to the cells transduced with the empty plasmid. J mtDNA content evaluation by qPCR. All data are means + SEM of six biological replicates and are normalized to the empty cells. K m.8344A > G mutation heteroplasmy evaluation by snapshot method. Values are expressed as means and SEM of three biological replicates. 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). Data are means ± SEM of three independent experiments (biological replicates). M Basal, ATP-linked and maximal respiration were calculated from OCR traces and reported in the graph. All values are means and SEM of three independent experiments (biological replicates). 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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