Table 1.
Sequence of primers for RT-PCR.
Fig 1.
COX deficiency in the CDs rats.
(A) COX activity normalized to citrate synthase (CS) activity in fibroblasts, liver, heart and pancreatic-islets of CDs and control rats. (B) COX activity per mg protein. (C) CS activity per mg protein. (D) Western-blot analysis of COX1. Values are mean ± S.E. of three independent experiments. *p<0.05, **p<0.01.
Fig 2.
(A) Representative confocal microscopy images of control (upper panel) and CDs fibroblasts (lower panel) stained with MitoTracker Red. (B) Quantification of MitoTracker Red fluorescence intensities per cell volume (n = 200 cells). Values are mean ± S.E. of three independent experiments. Scale bar, 10 μm. **p<0.01.
Fig 3.
Cell biomass, mitochondrial content, mitochondrial membrane potential, ATP content and ROS production.
Fibroblasts were grown in either DMEM-Glu or DMEM-Gal and examined as described under materials and methods. (A) Cell biomass. (B) Mitochondrial content measured by MTG. (C) Mitochondrial membrane potential. (D) Cellular ATP content. (E) ATP content per mitochondria. (F) Kinetics of ROS production. Results are shown as normalized to cell biomass measured by methylene blue (MB) and values are means ± S.E. RFU, relative fluorescence units; RLU, relative luminescence units. (n = 3). *p<0.05, **p<0.01.
Fig 4.
(A) Basal and maximal OCR levels. Basal OCR was measured before Oligomycin injection and maximum OCR after FCCP injection, subtracted by non-mitochondrial respiration (measurement after rotenone and Antimycin A injection). (B) Basal and maximal ECAR levels. Basal ECAR was measured before Oligomycin injection and maximal ECAR levels were measured after the injection of Oligomycin. (C) OCR/ECAR ratio under basal conditions. All OCR and ECAR values were normalized to cell biomass measured by methylene blue (MB). Analysis was performed in five replicate wells for each fibroblast line. Data are mean ± S.E.
Fig 5.
(A) Relative mRNA-expression of mitochondrial-biogenesis related genes. (B) Relative mRNA-expression of COX subunits. (C) Relative mRNA-expression of COX chaperones. (D) Western-blot analysis of pAMPK, pACC and PGC1-α. On the right panel, band intensity ratios relative to α-Tubulin are shown. Western blots images are representative of 3 independent experiments. Gene expression is shown as fold change of expression relative to control fibroblasts. Data are mean ± S.E. (n = 3). *p<0.05, **p<0.01.
Fig 6.
Western-blot analysis of LC3-I and LC3-II. Fibroblasts were grown under normal or starvation (starv) conditions in the presence or absence of autophagy inhibitor bafilomycin A1 (baf, 100 nM) for 4 h as described under materials and methods. Lower panel shows the band intensity ratio of LC3-II to LC3-I. Data represent mean ± S.E. of three independent experiments. *p<0.05.
Fig 7.
Co-staining with Mitotracker Red and anti-LC3 antibody in the presence or absence of autophagy inhibitor Bafilomycin A1. The upper panels shows the control fibroblasts and the lower panels the CDs fibroblasts. Below is shown quantitative analysis of fluorescence intensity (arbitrary units) of LC3 and MitoTracker Red co-localization per cell. Data represent mean ± S.E. of three independent experiments. Scale bar, 10 μm.