Immunoblotting. GAPDH served as a sample loading control. (D) Cell morphology and protein location of F-actin in H9C2 cells were analyzed by immunostaining. H9C2 cells on coverslips were either left untreated, treated with doxorubicin or pre-treated with quercetin prior to doxorubicin treatment before fixation and staining. F-actin was stained with phalloidin and nuclei were stained with DAPI. Each set of five fields were taken using the same exposure and images are representative of five different fields. In (B) (D), 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.our previous reports [20-22]. All primary antibodies used for expression validation were purchased from Genetex (Hsinchu, Taiwan).2D-DIGE, gel image analysis, protein staining, in-gel digestion and MALDI-TOF MS analysisThe detailed experimental PNPP molecular weight procedures have been described in our previous publications [23-25]. Notably, peaks in the mass range of m/z 800-3000 were used to generate a peptide mass fingerprint that was searched against the Swiss-Prot/TrEMBL database (released on August 2011) with 531473 entries using Mascot software v2.3.02 (Matrix Science, London, UK). The parameters used for Mascot search are listed: mouse; tryptic digest with a maximum of 1 missed cleavage; carbamidomethylation of cysteine, partial protein N-terminal acetylation, partial methionine oxidation and partial modification of glutamine to pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on significant MASCOT Mowse scores (p < PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 0.05), spectrum annotation and observed versus expected molecular weight and pI on 2-DE as well as at least 5 peptides in each identified protein.ResultsQuercetin facilitates cell survival and maintains cell morphology in doxorubicin-induced cell death in H9C2 cellsusing quercetin in concentrations from 50 to 200 M (Isovaleryl-Val-Val-Sta-Ala-Sta-OH price Figure 2A). Because excess ROS stress from doxorubicin-treated cardiomyocyte alters redox homeostasis and induces cell death, cell apoptosis was further detected using FACS. During cell apoptosis, phosphatidylserine is translocated to the outer surface of the plasma membrane, which has a high affinity to annexin V-FITC, and PI can penetrate the cell nucleus. As shown in Figure 2B, the apoptotic rate increased from 4.9 to 61.4 after doxorubicin treatment, whereas the apoptotic rate decreased to 9.5 after the H9C2 cells were pretreated with quercetin before doxorubicin treatment. In addition, the levels of the proteolytic enzymes, caspase 9 and caspase 3, were detected using immunoblotting in control, doxorubicin-treated and quercetin-pretreated H9C2 cells. Figure 2C indicates increased expression levels of the apoptosis factors for caspase 3 and caspase 9 after doxorubicin treatment. Quercetin protected the H9C2 cells from doxorubicininduced cell injury by inhibiting the expressions of caspase 3 and caspase 9. Additionally, immunostained images of F-actin indicated that doxorubicin treatment affected cytoskeletal protein reorganization, causing cell morphology alternation (Figure 2D). Therefore, quercetin pretreatment is essential to maintaining doxorubicin-induced morphological changes.2D-DIGE analysis of untreated and doxorubicin-treated H9C2 cells and quercetin pretreatment followed by doxorubicin treatmentTo evaluate the effect of doxorubicin on rat cardiomyocytes (H9C2), we exposed the cells to doxorubicin in a range of 0-1 M for 24 h in a ser.Immunoblotting. GAPDH served as a sample loading control. (D) Cell morphology and protein location of F-actin in H9C2 cells were analyzed by immunostaining. H9C2 cells on coverslips were either left untreated, treated with doxorubicin or pre-treated with quercetin prior to doxorubicin treatment before fixation and staining. F-actin was stained with phalloidin and nuclei were stained with DAPI. Each set of five fields were taken using the same exposure and images are representative of five different fields. In (B) (D), 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.our previous reports [20-22]. All primary antibodies used for expression validation were purchased from Genetex (Hsinchu, Taiwan).2D-DIGE, gel image analysis, protein staining, in-gel digestion and MALDI-TOF MS analysisThe detailed experimental procedures have been described in our previous publications [23-25]. Notably, peaks in the mass range of m/z 800-3000 were used to generate a peptide mass fingerprint that was searched against the Swiss-Prot/TrEMBL database (released on August 2011) with 531473 entries using Mascot software v2.3.02 (Matrix Science, London, UK). The parameters used for Mascot search are listed: mouse; tryptic digest with a maximum of 1 missed cleavage; carbamidomethylation of cysteine, partial protein N-terminal acetylation, partial methionine oxidation and partial modification of glutamine to pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on significant MASCOT Mowse scores (p < PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 0.05), spectrum annotation and observed versus expected molecular weight and pI on 2-DE as well as at least 5 peptides in each identified protein.ResultsQuercetin facilitates cell survival and maintains cell morphology in doxorubicin-induced cell death in H9C2 cellsusing quercetin in concentrations from 50 to 200 M (Figure 2A). Because excess ROS stress from doxorubicin-treated cardiomyocyte alters redox homeostasis and induces cell death, cell apoptosis was further detected using FACS. During cell apoptosis, phosphatidylserine is translocated to the outer surface of the plasma membrane, which has a high affinity to annexin V-FITC, and PI can penetrate the cell nucleus. As shown in Figure 2B, the apoptotic rate increased from 4.9 to 61.4 after doxorubicin treatment, whereas the apoptotic rate decreased to 9.5 after the H9C2 cells were pretreated with quercetin before doxorubicin treatment. In addition, the levels of the proteolytic enzymes, caspase 9 and caspase 3, were detected using immunoblotting in control, doxorubicin-treated and quercetin-pretreated H9C2 cells. Figure 2C indicates increased expression levels of the apoptosis factors for caspase 3 and caspase 9 after doxorubicin treatment. Quercetin protected the H9C2 cells from doxorubicininduced cell injury by inhibiting the expressions of caspase 3 and caspase 9. Additionally, immunostained images of F-actin indicated that doxorubicin treatment affected cytoskeletal protein reorganization, causing cell morphology alternation (Figure 2D). Therefore, quercetin pretreatment is essential to maintaining doxorubicin-induced morphological changes.2D-DIGE analysis of untreated and doxorubicin-treated H9C2 cells and quercetin pretreatment followed by doxorubicin treatmentTo evaluate the effect of doxorubicin on rat cardiomyocytes (H9C2), we exposed the cells to doxorubicin in a range of 0-1 M for 24 h in a ser.
