Fig. 3
Nicotinic acid (NA) treatment in MERRF cybrids and fibroblasts. Cells are treated with vehicle (DMSO) or 10 mM NA for 96 h. A, B OCR in cybrids A and fibroblasts B after NA treatment; OCR expressed as pmoles O2/min and normalized for protein content, under basal conditions and after injection of oligomycin (O), carbonyl cianide 4-(trifluoromethoxy) phenylhydrazone (FCCP; F), rotenone (R) and antimycin A (AA). In cybrids, data are means ± SD of two experiments, analyzing two biological replicates. In fibroblasts, data are means ± SD of three experiments for Wt-F and of two experiments for H-F; only one experiment could be performed in I-F due to spontaneous heteroplasmy shift in culturing cells. C, D Western blot of OXPHOS subunits in cybrids C and fibroblasts D; ACTIN was used as loading control. A representative blot is shown for each protein. E Densitometry of OXPHOS subunits content in cybrids. Data of four independent experiments (biological replicates) are expressed as means and SEM, and are normalized to untreated cells. F Densitometry of OXPHOS subunits content in fibroblasts. Data are means and SD of three biological replicate for Wt-F and of two biological replicates for H-F; only one experiment could be performed in I-F due to spontaneous heteroplasmy shift in culturing cells; all data are normalized to untreated cells. G mtDNA content evaluation by qPCR in cybrids. All values are expressed as means and SEM of three biological replicates and are normalized to untreated cells. H Heteroplasmy level evaluation by snapshot method in cybrids. Values are expressed as means and SEM of three biological replicates. I mtDNA content evaluation by qPCR in fibroblasts. Data are means and SD of three biological replicates for Wt-F and of two biological replicates for H-F; only one experiment could be performed in I-F due to heteroplasmy shift in culturing cells. J Heteroplasmy level evaluation by snapshot method in fibroblasts. H-F values are expressed as means and SD of two biological replicates. Statistical analyses were performed using unpaired two-tail T-test. P value: * p < 0.05, ** p < 0.01, *** p < 0.001