Fig 2: Inhibition of Notch 1 expression with Notch 1-specific-siRNA suppressed the activity of the Akt/mTOR signaling pathway in clear cell renal cell carcinoma 786-O cells. Expression of key components in the Akt/mTOR signaling pathway was evaluated by western blot analysis. (A) Inhibition of Notch 1 expression had no effect on total Akt expression, but inhibited the expression of p-Akt, p-mTOR and p-P70S6K in a dose-dependent manner. Quantitative determination of (B) Akt and p-Akt, and (C) p-mTOR and p-P70S6K expression following normalization by β-actin. Data are presented as the mean ± standard deviation of three independent western blot assays and presented as column diagrams. *P<0.05, **P<0.01 vs. control. siRNA, small interfering RNA; mTOR, mechanistic target of rapamycin; p-, phosphorylated-.

Fig 3: Activation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) contributes to the protective effect of Ang-(1-7) against HG-induced reduction of mitochondrial membrane potential (MMP). (A-H) MMP was detected with rhodamine 123 (Rh123) staining followed by photofluorography. (A) Control group. (B) H9c2 cells were exposed to 35 mM glucose for 24 h. (C) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose for 24 h. (D) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 1 μM A-779 for 24 h. (E) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 10 μM LY294002 for 24 h. (F) H9c2 cells were treated with 1 μM Ang-(1-7) for 24 h followed by 24-h culture. (G) H9c2 cells were treated with 1 μM A-779 for 24 h followed by 24-h culture. (H) H9c2 cells were treated with 10 μM LY294002 for 24 h followed by 24-h culture. (I) Quantitative analysis of the mean fluorescence intensity (MFI) of Rh123 in (A-H) using ImageJ 1.41o software. Data are shown as means ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.

Fig 4: Ang-(1-7) mitigates HG-induced dephosphorylation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) in H9c2 cells. H9c2 cells were treated for 24 h with 35 mM glucose alone, or co-treated with 1 μM Ang-(1-7) and 35 mM glucose, or co-treated with 35 mM glucose, 1 μM Ang-(1-7) and 1 μM A-779, or treated with 1 μM Ang-(1-7) alone, or 1 μM A-779 alone. The expression of PI3K and Akt were detected by western blot analysis. (A and C) Variations in the expression levels of (A) PI3K and (C) Akt in the indicated groups. (B and D) Densitometric analysis for the results in (A) and (C), respectively. Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.

Fig 5: Activation of phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) is involved in the protective effect of Ang-(1-7) against HG-induced apoptosis in H9c2 cells. (A-H) After the indicated treatments, cellular apoptosis was assessed by Hoechst 33258 staining followed by photofluorography. (A) Control group. (B) H9c2 cells were exposed to 35 mM glucose for 24 h. (C) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose for 24 h. (D) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 1 μM A-779 for 24 h. (E) H9c2 cells were co-treated with 1 μM Ang-(1-7) and 35 mM glucose in the presence of 10 μM LY294002 for 24 h. (F) H9c2 cells were treated with 1 μM Ang-(1-7) for 24 h followed by 24-h culture. (G) H9c2 cells were treated with 1 μM A-779 for 24 h followed by 24-h culture. (H) H9c2 cells were treated with 10 μM LY294002 for 24 h followed by 24-h culture. (I) The apoptosis rate was analysed with a Cell Counting Kit-8 and ImageJ 1.41o software. Data are presented as mean ± standard error of the mean (n=3). **P<0.01 compared with the control group; ##P<0.01 compared with the HG-treated group; ΔΔP<0.01 vs. the group co-treated with Ang-(1-7) and HG. Ang-(1-7), angiotensin-(1-7); HG, high glucose (35 mM); LY, LY294002.