Most relevant differences in terms of gene expression in H9c2 undifferentiated myoblasts vs RA-differentiated H9c2 cells.

<p>H9c2 cells were differentiated for 5 days in a low serum concentrated medium daily supplemented with RA. Both groups, undifferentiated and RA-differentiated cells, were collected and analyzed as described in Material and Methods section. FC>2 corresponds to, at least, two-fold up-regulation of the gene, FC<2 corresponds to, at least, two-fold down-regulation of the gene in differentiated cells with RA. Data correspond to four separate experiments and differences are also represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129303#pone.0129303.g002" target="_blank">Fig 2</a>. Abbreviation: Dif–genes related with differentiation process to a cardiac phenotype; CellSig–genes encoding proteins related with cell signaling pathways; Metabol—genes related with metabolic activity; Cellcycle—genes related with proliferation activity; Calcium–genes related with cellular calcium handling.</p><p>Most relevant differences in terms of gene expression in H9c2 undifferentiated myoblasts vs RA-differentiated H9c2 cells.</p

Metabolic and signaling pathways represented by the alternatively expressed gene transcripts in RA-differentiated cells.

<p>The pool of significantly up- and down-regulated genes in RA-differentiated cells was analyzed for the specific enrichment in any biochemical pathways using DAVID Bioinformatics Resources engine and KEGG-based database. Top 10 pathways showed significant enrichment with up-regulated and down-regulated genes in the differentiated cells are shown.</p><p>Metabolic and signaling pathways represented by the alternatively expressed gene transcripts in RA-differentiated cells.</p

Semi-quantification of selected proteins involved on calcium handling and metabolism by Western blotting.

<p>Protein content of pyruvate dehydrogenase, hexokinase II, Pink, SERCA2 and glutathione peroxidase 1 was measured by Western blotting as described in the materials and methods section. H9c2 myoblasts were differentiated by being cultured in 1% FBS medium supplemented with 1 μM RA and maintained for 5 days. Total extracts from both cellular groups were collected. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4 independent experiments (*) p<0.05 versus undifferentiated group.</p

Evaluation by Western blotting of selected proteins with probable involvement on the differentiation of H9c2 cells towards a cardiac-like cells.

<p>Cellular content of Creb (total and phosphorylated form), PI3K (Class III and p85), PTEN and PDK1were measured as described in the materials and methods section. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4 independent experiments (*) p<0.05 vs undifferentiated group.</p

Evaluation by Western blotting of selected proteins with probable involvement on the differentiation of H9c2 cells towards a cardiac-like cells.

<p>Cellular content of Akt, Gata 4, ERK1 (total and phosphorylated), and ERK2 (total and phosphorylated) were measured as described in the materials and methods section. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4 independent experiments (*) p<0.05 vs respective undifferentiated group.</p

Semi-quantification by Western blotting of selected proteins involved on mitochondrial function and metabolism.

<p>Protein content of PGC1α, TOM20, complex I NDUFS4 subunit, uncoupling protein 3, creatine kinase and lactate dehydrogenase was measured as described in the materials and methods section. H9c2 myoblasts were differentiated by being cultured in 1% FBS medium supplemented with 1 μM RA and maintained for 5 days. Total extracts from both cellular groups were collected. Ponceau labeling represents the loading control and was used to normalize data. Data represent the mean ± SEM of 4–5 independent experiments (*) p<0.05 versus respective undifferentiated group.</p