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

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

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

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

Blood plasma profile after DOX treatment.

#<p>For troponin I analysis, only 13 and 16 samples were measured in acute and sub-chronic protocol, respectively due to limitations in the analytical kit used.</p><p>Differences between treatment groups means within the same model were evaluated by Student’s t test, when assumptions were not achieved a Welch correction or the non-parametric Mann-Whitney test were applied (see Material and Methods for detailed information).</p>*<p>p≤0.05;</p>**<p>p≤0.01;</p>***<p>p≤0.001 vs saline group of the same model. List of abbreviations: CK – creatine kinase; TnI – troponin I; TRIG – triglycerides; CHOL – total cholesterol; AST – aspartate aminotransferase; ALT – alanine aminotransferase; TP – total serum proteins; LDH – lactate dehydrogenase; CREA – creatinine; UA – uric acid; BUN – blood urea nitrogen; SE – standard error.</p

Body and organs mass profile of animals subjected to DOX treatment protocols.

<p>Data refers to wet organ mass and its ratio to body mass was obtained dividing the organ mass over the respective total animal mass times 1000. The deceased DOX-treated rat and its matched control in the sub-chronic model were excluded from this analysis. Differences between treatment groups means within the same model were evaluated by Student’s t test (see Material and Methods for detailed information).</p>*<p>p≤0.05;</p>**<p>p≤0.01;</p>***<p>p≤0.001 vs saline group of the same treatment protocol. HM:BM – heart mass to body mass ratio; LM:BM – liver mass to body mass ratio; KM:BM – kidney mass to body mass ratio; SE – standard error.</p

Histological analysis of organs collected from rats treated with DOX.

<p>No notorious differences or hallmarks of DOX toxicity were found in the different tissues in both protocols. Panels represent HE photographs of random chosen tissues: hearts present minor cytoplasmatic vacuolization (Panel <b>B</b>) and cytoplasmatic dilatation (Panel <b>D</b>); liver usually show minor cytoplasmatic vacuolization (Panel <b>F</b> and <b>H</b>); no changes in kidneys (Panel <b>J</b> and <b>L</b>). Organs were fixed in Bouin’s solution, processed through standard histological procedures and stained with HE (for more information, see Material and Methods).</p

Echocardiogram parameters in the sub-chronic protocol.

<p>Differences between treatment groups were evaluated by non-parametric Mann-Whitney test due to their lack of normality (see Material and Methods for detailed information). IVS – interventricular septum; LPWT – left posterior wall thickness; LVDd – left ventricular diastolic dimension; LVDs – left ventricular systolic dimension; LVEF – left ventricular ejection fraction; FS – fraction shortening; AT s/d – arterial tension systole/diastole.</p