Journal: Cardiovascular diabetology

Article Title: Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis.

doi: 10.1186/s12933-023-01747-1

Figure Lengend Snippet: Fig. 2 Cardiac ASMase promotes NADPH oxidases and apoptosis in HFD mice. a Representative Western blot images of ASMase expression in the myocardium of HFD-induced mice with or without imipramine. b Quantitative analysis of ASMase expression, β-actin was used as a loading control. c ASMase activity was detected by assay kit. d Representative immumohistochemical staining of ceramide (scale bar: 100 μm). e Representative images of Oil Red staining in hearts and livers from four groups of mice (scale bar: 100 μm). f–i Representative Western blot images and quantitative analysis of NOX4, NOX2 and 4-HNE protein expressions in hearts of four groups. β-actin was used as a loading control. j Representative fluorescence images of TUNEL staining (left scale bar: 1000 μm; right scale bar: 50 μm). k Quantitative analysis of TUNEL-positive cells in myocardial tissues. Data were presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The membrane was blocked with 5% skim milk for 1 h, and then incubated with the prepared primary antibodies ASMase (3687S, 1:1000, Cell Signaling Technology), NOX2 (19013-1- AP, 1:3000, Proteintech Group), NOX4 (14347-1-AP, 1:3000, Proteintech Group), Cleaved-caspase-3 (9662S, 1:1000, Cell Signaling Technology), 4- Hydroxynonenal (ab46545, 1:3000, Abcam) and β-actin (BS6007M, 1:6000, Bioworlde) at 4 °C overnight.

Techniques: Western Blot, Expressing, Control, Activity Assay, Staining, Fluorescence, TUNEL Assay

Journal: Cardiovascular diabetology

Article Title: Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis.

doi: 10.1186/s12933-023-01747-1

Figure Lengend Snippet: 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

Article Snippet: The membrane was blocked with 5% skim milk for 1 h, and then incubated with the prepared primary antibodies ASMase (3687S, 1:1000, Cell Signaling Technology), NOX2 (19013-1- AP, 1:3000, Proteintech Group), NOX4 (14347-1-AP, 1:3000, Proteintech Group), Cleaved-caspase-3 (9662S, 1:1000, Cell Signaling Technology), 4- Hydroxynonenal (ab46545, 1:3000, Abcam) and β-actin (BS6007M, 1:6000, Bioworlde) at 4 °C overnight.

Techniques: Western Blot, Expressing, Control, Fluorescence, Staining, TUNEL Assay

Journal: Cardiovascular diabetology

Article Title: Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis.

doi: 10.1186/s12933-023-01747-1

Figure Lengend Snippet: Fig. 4 ASMase knockdown dampens high glucose, palmitate acid and ceramide induced oxidative stress and apoptosis in H9c2 cells. a Representative Western blot images of ASMaseexpression when H9c2 cells were posed to vehicle or pretransfected with siRNA ASMase (si-ASMase), scrambled siRNA (si-control) was used as control (n = 5). b–c Representative Western blot and quantitative analysis images of ASMase, NOX2 and NOX4 expression when H9c2 cells were treated with vehicle, glucose + PA, ceramide, siASMase, siASMase + glucose + PA, siASMase + ceramide, respectively. β-actin was used as a loading control. Three to five independent experiments were performed to calculate the means. d Quantitative analysis of NOX1, NOX2 and NOX4 mRNA expression by qPCR in H9c2 cardiomyocytes after transfected by si-ASMase. Four independent experiments were performed to calculate the means. e Representative fluorescence images of rhodamine-phalloidin staining to visualize cell size, scale bar:100 μm. f Quantitative analysis of pro-hypertrophic markers, Nppb and Myh7 mRNA expression by qPCR in H9c2 cardiomyocytes. Four independent experiments were performed to calculate the means. g–h 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. i Representative Western blot images of protein expression of cleaved caspase-3, β-actin was used as a loading control. j–k 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

Article Snippet: The membrane was blocked with 5% skim milk for 1 h, and then incubated with the prepared primary antibodies ASMase (3687S, 1:1000, Cell Signaling Technology), NOX2 (19013-1- AP, 1:3000, Proteintech Group), NOX4 (14347-1-AP, 1:3000, Proteintech Group), Cleaved-caspase-3 (9662S, 1:1000, Cell Signaling Technology), 4- Hydroxynonenal (ab46545, 1:3000, Abcam) and β-actin (BS6007M, 1:6000, Bioworlde) at 4 °C overnight.

Techniques: Knockdown, Western Blot, Control, Expressing, Transfection, Fluorescence, Staining, TUNEL Assay

Journal: Cardiovascular diabetology

Article Title: Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis.

doi: 10.1186/s12933-023-01747-1

Figure Lengend Snippet: Fig. 5 NOX4 activation is required for ASMase-mediated cardiac hypertrophy and apoptosis. a–b Representative Western blot images and quantitative analysis of protein expression of ASMase, NOX2, and NOX4 in six groups of H9c2 cells treated with vehicle, glucose + PA, ceramide, apocynin, apocynin + glucose + PA, apocynin + ceramide, respectively. β-actin was used as a loading control. 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

Article Snippet: The membrane was blocked with 5% skim milk for 1 h, and then incubated with the prepared primary antibodies ASMase (3687S, 1:1000, Cell Signaling Technology), NOX2 (19013-1- AP, 1:3000, Proteintech Group), NOX4 (14347-1-AP, 1:3000, Proteintech Group), Cleaved-caspase-3 (9662S, 1:1000, Cell Signaling Technology), 4- Hydroxynonenal (ab46545, 1:3000, Abcam) and β-actin (BS6007M, 1:6000, Bioworlde) at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Expressing, Control, Fluorescence, Staining, TUNEL Assay

Journal: Cardiovascular diabetology

Article Title: Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis.

doi: 10.1186/s12933-023-01747-1

Figure Lengend Snippet: Fig. 7 ASMase deficiency reduces NAPDH oxidase activity and apoptosis in HFD mice. a–d Representative Western blot images and densitometric analysis of ASMase, NOX2 and NOX4 protein levels in ASMaseMyh6KO and control mice treated with or without HFD. e Representative immumohistochemical staining of ceramide in myocardium of mice (scale bar:100 μm). f–g Representative fluorescence images and quantitative analysis of TUNEL staining (scale bar:100 μm). Data were presented as Mean ± SEM, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: The membrane was blocked with 5% skim milk for 1 h, and then incubated with the prepared primary antibodies ASMase (3687S, 1:1000, Cell Signaling Technology), NOX2 (19013-1- AP, 1:3000, Proteintech Group), NOX4 (14347-1-AP, 1:3000, Proteintech Group), Cleaved-caspase-3 (9662S, 1:1000, Cell Signaling Technology), 4- Hydroxynonenal (ab46545, 1:3000, Abcam) and β-actin (BS6007M, 1:6000, Bioworlde) at 4 °C overnight.

Techniques: Activity Assay, Western Blot, Control, Staining, Fluorescence, TUNEL Assay