Fig. 9
Effects of ADAM17 and ACE2 double knock-down and AMPK inhibitor on cardiac autophagy and apoptosis. a Representative Western blot images of protein expression of phosphorylated AMPK, AMPK, p62 and LC3 in six groups of NRCMs treated with vehicle, GP, GP + NC-siRNA, GP + ADAM17-siRNA, GP + ACE2-siRNA and GP + ADAM17-siRNA+ACE2-siRNA, respectively. b–d Quantitative analysis of p-AMPK/AMPK, p62 and LC3II/β-actin expression in six groups of NRCMs. Mean values were derived from six independent experiments. Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. the vehicle group; #P < 0.05, ##P < 0.01 vs. the GP + NC-siRNA group. e Representative Western blot images of Bax, Bcl2, and cleaved Caspase-3 expression in four groups of NRCMs treated with GP + NC-siRNA+vehicle, GP + ADAM17-siRNA+vehicle, GP + NC-siRNA+CQ and GP + ADAM17-siRNA+CQ, respectively. f, g Quantitative analysis of Bax/Bcl2, and cleaved Caspase-3 expression in four groups of NRCMs. Five independent experiments were conducted to derive the means. Data were expressed as mean ± SEM. **P < 0.01, ***P < 0.001 vs. the GP + NC-siRNA+vehicle group; ###P < 0.001 vs. the GP + A17-siRNA group+vehicle group. h Representative Western blot images of Bax, Bcl2, p-AMPK, AMPK, p62 and LC3 expression in four groups of NRCMs treated with GP + NC-siRNA+vehicle, GP + ADAM17-siRNA+vehicle, GP + NC-siRNA+dorsomorphin, GP + ADAM17-siRNA+dorsomorphin, respectively. i–l Quantitative analysis of Bax/Bcl2, p-AMPK/AMPK, p62 and LC3II/β-actin expression in four groups of NRCMs. Mean values were derived from six independent experiments. Data were shown as mean ± SEM. **P < 0.01, ***P < 0.001 vs. the GP + NC-siRNA+vehicle group; ##P < 0.01, ###P < 0.001 vs. the GP + A17-siRNA group+vehicle group. m Representative Western blot images of HIF-1α and ADAM17 expression in four groups of NRCMs treated with vehicle+NC-siRNA, GP + NC-siRNA, vehicle+ HIF-1α-siRNA and GP + HIF-1α-siRNA, respectively. n, o Quantitative analysis of HIF-1α and ADAM17 expression in four groups of NRCMs. Five independent experiments were performed to calculate the means. Data were presented as mean ± SEM. **P < 0.01, ***P < 0.001 vs. the vehicle+NC-siRNA group; ##P < 0.01 vs. the GP + NC-siRNA group. p, q Representative Western blot images and analysis of ADRA1A protein expression in the myocardium of four groups of mice, n = 5. **P < 0.01 vs. the A17fl/fl control group; ##P < 0.01 vs. the A17fl/fl DM group. r, s Representative Western blot images and quantitative analysis of ADRA1A protein expression in NRCMs treated with vehicle, GP, GP + NC-siRNA, GP + ADAM17-siRNA, GP + ACE2-siRNA and GP + ADAM17-siRNA+ACE2-siRNA, respectively. Five independent experiments were conducted to derive the means. Data were expressed as mean ± SEM. *P < 0.05 vs. the vehicle group; #P < 0.05, ##P < 0.01 vs. the GP + NC-siRNA group. t and u Representative fluorescence images of JC-1 staining in NRCMs treated as above (scale bar: 20 μm). Quantitative analysis of JC-1 (JC-1 aggregates/JC-1 monomers) in NRCMs. Six independent experiments were performed to derive the mean values. Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. the vehicle group; #P < 0.05, ##P < 0.01 vs. the GP + NC-siRNA group
