Um-free medium. After exposure to doxorubicin, dose dependent loss of cellUm-free medium. After exposure to

Um-free medium. After exposure to doxorubicin, dose dependent loss of cell
Um-free medium. After exposure to doxorubicin, dose dependent loss of cell viabilities was observed in the H9C2 cells in 3 independent experiments using MTT assays (Figure 1). At a concentration of 0.45 M, a significant loss (50 ) of cell viability was detected after 24 h. To verify the role of quercetin regarding the recovery of doxorubicin-induced cardiomyopathy, we investigated the changes in cell viability in the H9C2 cells incubated in 0 M, 50 M, 100 M, 150 M and 200 M quercetin for 4 h, followed by 24 h-exposure to 0.45 M doxorubicin. Our results demonstrated that cell viability was significantly improvedTo fully understand the roles of doxorubicin and quercetin pretreatment in H9C2 cells, lysates of cells untreated, treated with doxorubicin, or treated with doxorubicin after pretreatment of quercetin, were subjected to 2D-DIGE analysis. The results of the 2D-DIGE analysis and DeCyder processing identified 2156 protein spots, and 73 proteins exhibited differential expression ( 1.5 fold or -1.5 fold; p < 0.05) among the 3 conditions (Figure 3). Additional file 1: Table S1 shows the 73 proteins that were identified using MALDI-TOF MS, and 31 of the 73 identifiedChen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 5 ofAGel 1 Gel 2 Gel 3 Gel 4 GelCy2 (100 pool pool pool pool poolg) Cy3 (100 g) Cy5 (100 g) Ctrl Dox Dox Dox+Que Dox+Que Ctrl Ctrl Dox+Que Dox+Que DoxpH = 3 pH = 10 pH = 3 pH =BMWpH =pH =ControlDoxpH = 10 pH = 3 pH =Que + DoxCMWpH =Green: ControlRed: DoxGreen: ControlRed: Que + DoxpH =DMWpH =Figure 3 (See legend on next page.)Chen et al. Journal of Biomedical Science 2013, 20:95 http://www.jbiomedsci.com/content/20/1/Page 6 of(See figure on previous page.) Figure 3 2D-DIGE analysis of H9C2 cells in response to doxorubicin treatment and pre-treatment with quercetin. (A) Samples arrangement for a triplicate 2D-DIGE experiment. (B) Protein samples (100 g each) were labeled with Cy-dyes and separated using 24 cm, pH 3-10 non-linear IPG strips. 2D-DIGE images of the protein samples from H9C2 cells in response to doxorubicin treatment and pre-treatment with quercetin at appropriate excitation and emission wavelengths were shown as well as overlaid pseudo-colored images processed with ImageQuant Tool (GE Healthcare) (C). (D) Protein samples (100 g each) purified from total cell lysates were labeled with Cy-dyes and separated using 24 cm, pH 3-10 non-linear IPG strips. The differentially expressed protein features were annotated with spot numbers. In this 2D-DIGE experiment, H9C2 cells were untreated, 0.45 M of doxorubicin for 24 h, or 100 M of quercetin for 4 h followed by 0.45 M of doxorubicin for 24 h.protein spots that displayed doxorubicin-dependent alteration can be reversed by pretreating with quercetin (Additional file 1: Table S1 and Additional file 2). For example, the 78 kDa glucose-regulated protein (GRP-78) (No.444) was up-regulated (1.63 fold) in the doxorubicintreated cells, whereas quercetin reduced the overexpression of doxorubicin-treated GRP-78 (-1.85 fold). Theresult suggested that the protective mechanisms of quercetin significantly altered the levels of chaperone proteins during doxorubicin-treatment in the cardiomyocytes. Figure 4A shows the functional distribution of the identified proteins from the 2D-DIGE results. Most PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28151467 of the proteins identified using MALDI-TOF MS were associated with the cytoskeletal element and cell migrationABFigure 4 DihexaMedChemExpress Dihexa Percentage of total diff.