Fig. 3

High glucose, palmitate acid and ceramide positively activate ASMase and induce oxidative stress and apoptosis in H9c2 cells. a Representative Western blot images of protein expression of ASMase, NOX2, and NOX4 in six groups of H9c2 cells treated with vehicle, glucose + PA, ceramide, imipramine, imipramine + glucose + PA, imipramine + ceramide, respectively. β-actin was used as a loading control. b Quantitative analysis of ASMase, NOX2, and NOX4 expression in H9c2 cells treated as above. Three independent experiments were performed to calculate the means. c Quantitative analysis of NOX1, NOX2 and NOX4 mRNA expression by qPCR in H9c2 cardiomyocytes. Five independent experiments were performed to calculate the means. d Representative fluorescence images of rhodamine-phalloidin staining to visualize cell size, scale bar: 100 μm. e Quantitative analysis of pro-hypertrophic markers, Nppb and Myh7 mRNA expression by qPCR in H9c2 cardiomyocytes. Five independent experiments were performed to calculate the means. f–g Representative fluorescence images and quantitative analysis of DCFH-DA staining for ROS measurement in H9c2 cells, scale bar:100 μm. Three independent experiments were performed to calculate the means. h Representative Western blot images of protein expression of cleaved caspase-3, β-actin was used as a loading control. i–j Representative fluorescence images and quantitative analysis of TUNEL staining, scale bar: 100 μm. Five independent experiments were performed to calculate the means. Data were presented as mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001