Effect of PGC-1α-overexpression on mitochondrial COX activity, mitochondrial to nuclear DNA ratio and oxidation of metabolic substrates in cultured muscle cells.

<p>Cultured muscle cells were transduced with Ad-GFP or Ad-PGC-1α adenoviruses. (A) Cytochemical staining of COX activity was performed in cultured skm cells that had been transduced with Ad-GFP (a, b and c) or Ad-PGC-1α (d, e and f). Images were obtained with a camera (a,b,d,e) or with an inverted microscope at 800× magnification (c,f). Images from two independent experiments performed in duplicate were quantitatively analyzed and the data expressed as percentages of the values in the controls: means ± SEM are shown on the graph. The significance of the difference is *p<0.005. (B) Cells were harvested and total DNA isolated to measure the ratio of mitochondrial DNA (mtDNA) content to nuclear DNA (nDNA) content. Data are expressed as a percentage of control. Data are means ± SEM from two experiments performed in triplicate. (C–E) Cells were then incubated for 4 h with: (C) 0.5 mM [1-¹⁴C]-palmitate (2.8 µCi/µmol), (D) 10 mM [U-¹⁴C]-glucose (0.21 µCi/µmol) and (E) 2 mM [U-¹⁴C]-lactate (0.6 µCi/µmol). Production of ¹⁴CO₂ was subsequently quantified. Data are expressed as means ± SEM from two experiments performed in sextuplicate. The significance of the difference is *p<0.005 and **p<0.001.</p

Upregulation of IL-8 and FITM1 protein content by PGC-1α-overexpression in cultured skm cells.

<p>Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α. In cell extracts, (A) ELISA assay for IL-8 and (B) immunoblot analysis of FITM1 were performed. (B) For FITM1, α-actinin was used as loading control; a representative image is shown and bands were quantified. (A and B) Data are means ± SEM from three experiments performed in triplicate. Significance of differences versus cells treated with Ad-GFP: *p<0.05.</p

Gene ontology annotation of upregulated genes in response to PGC-1α-overexpression in cultured muscle cells.

<p>List of relevant GO annotated from the set of upregulated transcripts with an absolute FC>1.5 and p<0.01 in response to PGC-1α-overexpression. Only GOs with more than one gene observed are shown. The first column lists the GO term identifier, the second the GO term description, the third the number of genes that are annotated with this GO term, and the fourth the false discovery rate adjusted p value of that term.</p

Differentially expressed genes in response to PGC-1α-overexpression in cultured muscle cells.

<p>Microarray analysis was performed on six cultured skm cell samples from three independent experiments, three transduced with Ad-GFP and three with Ad-PGC-1α adenovirus. The table lists the 53 regulated gene transcripts selected on the basis of the fold change (FC) and ordered by the magnitude of the absolute FC. Replicated genes, which occurred more than once in the microarray, were filtered: those appearing first in the list were arbitrarily chosen. The first column lists the NCBI Reference Sequence (RefSeq) accession number of the transcripts, the second the gene symbol, the third the description of the gene, the fourth the FC (with a positive symbol for upregulated genes and a negative symbol for downregulated ones) and the fifth the significance of differences as estimated by a paired t-test.</p

Effect of PGC-1α-overexpression on lipid droplets and triglyceride accumulation in cultured muscle cells.

<p>Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α and incubated with 0.5 mM oleate for 16 h. (A,B,C) Cells were fixed and then stained with Nile Red. (A) Representative lipid droplet micrographs obtained via confocal microscopy are shown. White bars represent 5 µm. (B) The number of lipid droplets per cell area and (C) the lipid droplet mean area values were quantified. Data are means ± SEM of (B) four cells or (C) at least 414 droplets. (D) Triglyceride content was measured. Data are means ± SEM of three experiments performed in quadruplicate. (B,C,D) The significance of the differences versus cells treated with Ad-GFP is *p<0.05 and **p<0.01.</p

Effect of PGC-1α-overexpression on glycogen metabolism and glucose uptake in cultured muscle cells.

<p>Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α. (A) Cells were incubated with 10 mM [U-¹⁴C]-glucose (0.10 µCi/µmol) for 18 h and then harvested to asses glucose incorporation into glycogen. Data are means ± SEM from two experiments performed in triplicate. (B) Glucose uptake was measured using 0.5 mM 2-deoxy-D-[³H]glucose (0.5 µCi/well). Data are means ± SEM from two experiments performed in triplicate. (C,D) Cells were harvested to measure (C) glycogen synthase and (D) glycogen phosphorylase activity. Data are means ± SEM from three experiments performed in quadruplicate. The significance of differences versus cells treated with control adenoviruses is indicated as follows: *p<0.05 and **p<0.01.</p

Genes validated for differential expression in PGC-1α-overexpressing cultured muscle cells.

<p>List of genes with changed expression levels in response to PGC-1α overexpression in cultured muscle cells as validated by RT and real-time PCR and ordered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029985#pone-0029985-t001" target="_blank">Table 1</a>. For each gene, the FC in gene expression was calculated from the mean values in PGC-1α-cells versus control cells from six independent experiments performed in triplicate. FC has a positive symbol for upregulated genes and a negative symbol for downregulated genes. All columns are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029985#pone-0029985-t001" target="_blank">Table 1</a>.</p

Genes regulated by both PGC-1α-overexpression in cultured skm cells and skm cultures in comparison to skm tissue.

<p>Heat map including the genes (Gene symbol/Gene ID for Entrez Gene) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029985#pone-0029985-t001" target="_blank">Table 1</a> (PGC-1α- versus control-skm cells) that were also found to be differentially expressed after human skm culture according to Table S2 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029985#pone.0029985-Raymond1" target="_blank">[25]</a> (cultured skm cells versus skm tissue). Each cell displays the absolute fold change in the corresponding comparison and is filled according to the color gradient shown at the top (green and red for down and upregulated genes, respectively).</p

Cell adhesion assay.

<p>Adherence of UCMD fibroblasts (n = 7) relative to control fibroblasts (n = 7) for fibronectin, vitronectin, laminin and collagen type I (experiments were performed in triplicate). To normalize adhesion values between experiments, we expressed the results as a ratio between the absorbance values for collagen type IV (which was the substrate that showed the smallest variability between individual cultures and experiments, data not shown) and each ECM protein, (student t-test * p < 0.05).</p

Ingenuity Pathway Analysis.

<p>Graphic representation of the network “cell cycle, skeletal and muscular system development”. Nodes represent genes and lines show the relationship between genes. The intensity of the node color indicates the degree of the up-regulation (red) or down-regulation (green) of significant genes in the P-C comparison. Non-color nodes are added by the tool. For a detailed legend refer to <a href="http://ingenuity.force.com/ipa/articles/Feature_Description/Legend" target="_blank">http://ingenuity.force.com/ipa/articles/Feature_Description/Legend</a>.</p