(Pro)renin receptor involves in myocardial fibrosis and oxidative stress in diabetic cardiomyopathy via the PRR–YAP pathway

Figures

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  Figure 1. (Pro)renin receptor and YAP expression in rats. (A) Representative immunostaining of PRR protein expression 2 weeks and 4 weeks after STZ intraperitoneal injection. (B) Quantitative analysis of the results shown in (A). (C) Representative immunostaining of YAP protein expression 2 weeks and 4 weeks after STZ intraperitoneal injection. The results are calculated by counting positive expression area intensity and totally available tissue area size, and then the final result is the ratio of positive area intensity and totally available tissue area size. (D) Quantitative analysis of the results shown in (C). (E) PRR mRNA expression. (F) PRR protein expression in the 3 different groups. Figure (F) is cropped picture, omitting the repeated blots from the same group. The full-length blots/gel is complemented in supplementary figure 1. (G) Quantitative analysis of the results shown in (F). (I) Representative immunostaining of YAP protein expression in the 3 groups. (H) Quantitative analysis of the results shown in (I). (scale bar = 20 μm, magnification × 40). **p < 0.01 compared with the DCM group and Ad-EGFP group; ##p < 0.01 compared with the control group. In this part, each group had…

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  Figure 2. TGF-β, CTGF and fibronectin expression in the 3 groups and SOD and, MDA levels and NADPH oxidase activity in the 3 groups. (A) Immunostaining of TGF-β, (B) Quantitative analysis of the results shown in (A). (C) Immunostaining of CTGF, (D) Quantitative analysis of the results shown in (C). (E) Immunostaining of fibronectin. (F) Quantitative analysis of the results shown in (E). (G) SOD levels in the 3 groups. (H) MDA levels in the 3 groups. (I) NADPH oxidase activity in the DCM group, Ad-EGFP group, and Ad-PRR group. *p < 0.05, **p < 0.01 compared with the DCM group and Ad-EGFP group. Scale bar = 20 μm, magnification × 40. In 4 weeks after removing dead rats, the other of rats incorporate into analysis, in DCM group had 11 rats, Ad-PRR group had 11 rats and Ad-EGFP group had 12 rats finally.

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  Figure 3. (Pro)renin receptor and YAP expression in cardiac fibroblasts. (A) Immunofluorescence showing PRR protein expression in the cardiac fibroblasts (CFs) in the control group, high permeability group and high glucose group. (B) Quantitative analysis of the results shown in (A). (C) Immunofluorescence showing YAP protein expression in the cardiac fibroblasts (CFs) in the control group, high permeability group and high glucose group. (D) Quantitative analysis of the results shown in (C). (E) Immunofluorescence showing YAP protein expression in the cardiac fibroblasts (CFs) in the high glucose group, Ad-EGFP group, and Ad-PRR group. (F) Quantitative analysis of the results shown in (E). **p < 0.01 compared with the control group and high permeability group. ##p < 0.01 compared with the high glucose group and Ad-EGFP group.

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  Figure 4. Smad3 and CTGF protein expression; TGF-β, collagen I and fibronectin expression and ROS production in cardiac fibroblasts (CFs) from the 3 groups. (A) Western blot showing Smad3 protein expression. Figure (A) is cropped picture, omitting the repeated blots from the same group. The full-length blots/gel is complemented in supplementary figure 4. (B) Quantitative analysis of the results shown in (A). (C) Western blot showing CTGF protein expression. (D) Quantitative analysis of the results shown in (C). (E–G) ELISA of TGF-β, collagen I, and fibronectin protein expression. (H) DHE showing ROS production in the 3 groups. (I) Quantitative analysis of the results shown in (H). **p < 0.01 compared with the Ad-EGFP group, #p < 0.05, ##p < 0.01 compared with the Ad-PRR group.

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  Figure 5. PRR and YAP protein expression, fibrosis and ROS production after PRR RNAi silencing. (A): Representative PRR protein expression after three types of Ad-PRR-shRNA transfection. Figure (A) is cropped picture, omitting the repeated blots from the same group. The full-length blots/gel is complemented in supplementary figure 5. (B) Quantitative analysis of the results shown in (A). (C) Immunofluorescence showing YAP protein expression in the cardiac fibroblasts (CFs) in the high glucose group, Ad-SC-shRNA group, and Ad-PRR-shRNA group. (D) Quantitative analysis of the results shown in (C). (E–H) PRR and YAP protein expression after PRR RNAi silencing. (E) PRR protein expression after PRR RNAi silencing in the 3 groups. (G) Quantitative analysis of the results shown in (E), **p < 0.01 compared with the Ad-SC-shRNA group and DCM group. (F) YAP protein expression after PRR RNAi silencing in the 3 groups. (H) Quantitative analysis of the results shown in (F), **p < 0.01 compared with the Ad-SC-shRNA group and DCM group. In this part, each group had n = 20 rats at beginning, in 2 weeks took 8 rats from each group for analysis. In 4 weeks after removing dead rats, the other of rats…

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  Figure 6. YAP, Smad3, CTGF and fibronectin expression in the 3 groups and SOD and, MDA levels and NADPH oxidase activity in the 3 groups. (A) YAP protein expression by Western blot. Figure (A) is cropped picture, omitting the repeated blots from the same group. The full-length blots/gel is complemented in supplementary figure 6. (B) Quantitative analysis of the results shown in (A). (C) Smad3 protein expression by Western blot. (D) Quantitative analysis of the results shown in (C). (E) Immunostaining of CTGF, (F) Quantitative analysis of the results shown in (E). (G) Immunostaining of fibronectin. (H) Quantitative analysis of the results shown in (G). (I) SOD levels in the 3 groups. (J) MDA levels in the 3 groups (K) NADPH oxidase activity in the DCM group, Ad-SC-shRNA group, and Ad-PRR-shRNA group. *p < 0.05, **p < 0.01 compared with the DCM group (n = 11) and LV-SC-shRNA group (n = 11). Scale bar = 20 μm, magnification × 40. In this part, each group had n = 20 rats at beginning, in 2 weeks took 8 rats from each group for analysis. In 4 weeks after removing dead rats, the other of rats incorporate into analysis, in DCM group had 11 rats, LV-SC-shRNA group had 11 rats and LV-YAP-shRNA…