Fig 2: Analysis of mitochondrial proteins.A, twenty micrograms of total cellular proteins from various cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with 11 mtDNA encoding polypeptides in mutant and control cells with GAPDH as a loading control. B, quantification of mitochondrial protein levels. Average relative ND1, ND3, ND4, ND5, ND6, CO1, CO2, CO3, ATP6, ATP8, and CYTB content per cell, normalized to the average content per cell of GAPDH in three mutant cell lines carrying the m.5587A>G mutation and three control cell lines lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell line. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 3.

Fig 3: SAA promotes mitochondrial biogenesis in 3T3-L1 adipocytes cells. 3T3-L1 cells were incubated with 10 nM SAA for 24 h. (a) Mitochondrial-to-nuclear DNA (mtDNA/nDNA) was determined by qRT-PCR analysis. Data are expressed as mean ± SEM of three independent measurements; (b) Mitochondrial mass was determined by staining with MitoTracker Green dye. Data are expressed as mean ± SEM of three independent measurements; (c) Morphometric analysis of surface area and density of mitochondria under the electron microscope. Data are expressed as mean ± SEM of data from six cells; (d) Representative illustrations of mitochondrial profiles under the electron microscope (magnification, ×40,000); (e) Protein levels of oxidative phosphorylation complex III (CYTB) and complex IV (MTCO1) were determined by Western blot analysis. Data are expressed as mean ± SEM of three independent measurements. *P < 0.05, **P < 0.01 vs. non-treated group.

Fig 4: Western blot analysis of mitochondrial proteins. (A) Analysis of mtDNA encoding proteins. Five micrograms of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies specific for ND1, ND2, ND4, ND5, ND6, CO2, CYTB and ATP8 and with Tom20 as a loading control, respectively. (B) Quantification of total mitochondrial protein levels. The average levels of 8 mitochondrial proteins in mutant and control cell lines were determined as described elsewhere (29,43). (C) Quantification of 8 polypeptides. The levels of ND1, ND2, ND4, ND5, ND6, CO2, CYTB and ATP8 in mutant and control cell lines were determined as described elsewhere (29,43). (D) Analysis of five OXPHOS subunits encoded by mtDNA and nuclear genes. Five micrograms of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibody cocktail specific for subunits (ATP5A, UQCRC2, SDHB, CO2 and NDUFB8) of each OXPHOS complex and with TOM20 as a loading control. Graph details and symbols are explained in the legend to Figure 3.

Fig 5: Western blot analysis of mitochondrial proteins. (A) Five micrograms of total mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with nine respiratory complex subunits in mutant, control cybrid cell lines and HUVECs with TOM20 as a loading control. ND1, ND3, ND4, ND5 and ND6, indicate subunits 1, 3, 4, 5 and 6 of the reduced nicotinamide-adenine dinucleotide dehydrogenase; CYTB, apocytochrome b; CO2, subunit II of cytochrome c oxidase; ATP6 and ATP8, subunit 6 and 8 of the H+-ATPase. (B) Quantification of total mitochondrial protein levels. The levels of mitochondrial proteins in HUVEC and six cybrid cell lines were determined as described elsewhere (22–24). The values for the mutant cybrid cell lines are expressed as percentages of the values for the control cell lines. The calculations were based on three independent determinations. (C) Quantification of levels of 9 polypeptides. The levels of ND1, ND3, ND4, ND5, ND6, CYTB, CO2, ATP6 and ATP8 in HUVEC and six cybrid cell lines were determined as described elsewhere (22–24). Graph details and symbols are explained in the legend to Figure 3.