miRNAs expression analysis.

<p>Real-time PCR was used to measure relative expression of miR-181a and miR-30c in skeletal muscle (A) and fibroblasts (B) from UCMD patients and in serum samples from UCMD, BM and DMD patients for miR-181a (C) and miR-30c (D). miRNA expression level was normalized against U6 miRNA. Results were calculated relative to control samples and are represented as mean and standard error.</p

Integrin-α3 expression in collagen VI deficient fibroblasts.

<p>A. Immunofluorescence for integrin-α3 in confluent fibroblast cultures. B. Representative western blot analysis. The intensity of the bands corresponding to integrin-α3 (ITGA3) was quantified by densitometry using α-tubulin (A-TUB) as a loading control and expressed as arbitrary units relative to the control samples.</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

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

Co-variance analysis was used to plot the expression values of a selection of genes of interest in untreated patient cells (P), control cells (C) and patient cells treated with AA (P_AA).

<p>Median and ranges are represented. A. HLA-DRA, B. COL1A1, C. TNC, D. TNX.</p

Extracellular matrix gene correlation network.

<p>This network represents Pearson correlations (R >0.8, p-value<0.005) computed considering expression values of genes of interest according to the microarray data and using Cytoscape tool. We selected those genes on the ECM-receptor interaction KEGG pathway and COL6A genes. Continuous lines represent positive correlations and discontinuous lines negative ones. Those correlations that are significant in patients´cells only are represented in black lines. Orange lines represent those correlations that are present in both patients and control cells but are of different sign (positive or negative) whereas those that have the same sign are represented by lilac lines. Red lines around gene symbols represent significantly over-expressed genes and green lines those that were under-expressed in patients´ fibroblasts relative to control fibroblasts.</p