Angiotensin II-induced muscle atrophy via PPARγ suppression is mediated by miR-29b

Figure 1. miR-29b inhibition ameliorates AngII-induced muscle atrophy in vitro (A) Immunofluorescent staining for C2C12 myotubes followed by determination of myotube diameter when C2C12 myotubes were transfected with negative control inhibitor (NC-1) and miR-29b inhibitor (29b-I) in AngII-induced muscle atrophy model. Scale bar: 50 μm. n = 4 per group. (B) Quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expressions when C2C12 myotubes were transfected with NC-1 and 29b-I in AngII-induced muscle atrophy model. n = 3, 4, 3. (C) Western blot analysis for the AKT/FOXO3A/mTOR pathway (AKT, FOXO3A, mTOR, P70S6K, 4EBP1) when C2C12 myotubes were transfected with NC-1 and 29b-I in AngII-induced muscle atrophy model. n = 3 per group. Data are shown as mean ± SD. ∗p < 0.05. ∗∗p < 0.01. See also Figure S1.

Figure 2. miR-29b inhibition ameliorates AngII-induced muscle atrophy in vivo (A) Quantitative real-time PCR analysis of miR-29b expression when mice were treated with Fugw (control) and miR-29b sponge in AngII-induced muscle atrophy. n = 4, 4, 5, 5. (B) Gastrocnemius weight from control and AngII mice treated with Fugw or miR-29b sponge. n = 5, 6, 5, 6. (C) The grip strength of right hindlimb from control and AngII mice treated with Fugw or miR-29b sponge. n = 4, 4, 5, 4. (D) Quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expression when control and AngII mice treated with Fugw or miR-29b sponge. n = 5–6 per group. (E) Wheat germ agglutinin (WGA) staining was performed to quantify muscle fiber cross-sectional area (CSA) from control and AngII mice treated with Fugw or miR-29b sponge. Scale bar: 20 μm. n = 5 per group. (F) TUNEL staining was performed to quantify apoptosis from control and AngII mice treated with Fugw or miR-29b sponge. Scale bar: 20 μm. n = 5, 5, 4, 5. (G) Western blot analysis for the ratio Bax/Bcl2, Cleaved-caspase 3/Caspase 3 when control and AngII mice treated with Fugw or miR-29b sponge. n = 3 per group. Data are shown as mean ± SD. ∗p < 0.05. ∗∗p <…

Figure 3. miR-29b mediates AngII-induced muscle atrophy by targeting Yin Yang 1 (YY1) (A) TargetScan and luciferase assay analysis of the direct interact with YY1 and miR-29b. n = 6 per group. (B) Quantitative real-time PCR analysis of Yy1 expression in C2C12 myotubes transfected with miR-29b mimic. n = 6 per group. (C) Quantitative real-time PCR analysis of Yy1 expression in C2C12 myotubes transfected with miR-29b inhibitor. n = 6 per group. (D) Western blot analysis of YY1 expression in C2C12 myotubes transfected with miR-29b mimic. n = 3 per group. (E) Western blot analysis of YY1 expression in C2C12 myotubes transfected with miR-29b inhibitor. n = 3 per group. (F) Western blot analysis of YY1 expression in C2C12 myotubes treated with AngII. n = 3 per group. (G) Western blot analysis of YY1 expression in the gastrocnemius from control and AngII treated mice. n = 3 per group. (H) Immunofluorescent staining for C2C12 myotubes and quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expression showed that overexpression of YY1 reduced muscle atrophy induced by AngII. n = 4 per group for staining. n = 3–4 for quantitative real-time PCR. Scale bar: 100 μm. (I) Immunofluorescent…

Figure 4. PPARγ acts as an upstream regulator of miR-29b (A) Schematic illustration of the binding sites of PPARγ in the promoter structures of miR-29b: the position of miR-29b-1: chr6:31221817-31221836 [−]; the position of miR-29b-2: chr1:195018028-195018047 [+]. (B) ChIP-PCR assay showed that PPARγ could bind to the promotor of miR-29b. n = 3 per group. (C) Luciferase assay analysis of the regulation function of PPARγ and miR-29b-1. n = 6 per group. (D) Luciferase assay analysis of the regulation function of PPARγ and miR-29b-2. n = 6 per group. (E) Quantitative real-time PCR analysis of miR-29b expression when C2C12 myotubes were transfected with sh-scramble and PPARγ shRNAs. n = 5, 7, 7. (F) Quantitative real-time PCR analysis of miR-29b expression when C2C12 myotubes were transfected with Fugw and PPARγ overexpression lentivirus. n = 4 per group. (G) Western blot analysis of PPARγ protein level in AngII-induced muscle atrophy in vitro. n = 3 per group. (H) Western blot analysis of PPARγ protein level in AngII-induced muscle atrophy in vivo. n = 3 per group. Data are shown as mean ± SD. ∗p < 0.05. ∗∗p < 0.01. See also Figure S4.

Figure 5. PPARγ inhibition with shRNAs promotes muscle atrophy in vitro (A) Immunofluorescent staining for C2C12 myotubes followed by determination of myotube diameter in C2C12 myotubes were treated with PPARγ shRNAs. Scale bar: 100 μm. n = 4 per group. (B) Quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expressions when C2C12 myotubes were treated with PPARγ shRNAs. n = 6, 4, 5–6. (C) Western blot analysis for the AKT/FOXO3A/mTOR pathway (AKT, FOXO3A, mTOR, P70S6K, 4EBP1) when C2C12 myotubes were treated with PPARγ shRNAs. n = 4 per group. Data are shown as mean ± SD. Sh-PPARγ-A and Sh-PPARγ-B group are compared with sh-scramble group. ∗p < 0.05. ∗∗p < 0.01. See also Figure S5.

Figure 6. Overexpression of PPARγ ameliorates AngII-induced muscle atrophy in vitro (A) Immunofluorescent staining for C2C12 myotubes followed by determination of myotube diameter and quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expression when myotubes were transfected with Fugw and PPARγ overexpression lentivirus in AngII-induced muscle atrophy model. Scale bar: 100 μm. n = 4 for staining and n = 6, 5, 5, 5 for quantitative real-time PCR. (B) Immunofluorescent staining for C2C12 myotubes followed by determination of myotube diameter and quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expression when myotubes were treated with PPARγ agonist rosiglitazone in AngII-induced muscle atrophy model. Ros, rosiglitazone. Scale bar: 100 μm. n = 3 for staining and n = 6 for quantitative real-time PCR. Data are shown as mean ± SD. ∗p < 0.05. ∗∗p < 0.01. See also Figure S6.

Figure 7. PPARγ inhibition promotes muscle atrophy in vivo (A) Gastrocnemius muscle morphology and weight in sh-scramble- and PPARγ shRNA-treated mice. Scale bar: 1 cm. n = 6. (B) The grip strength of right hindlimb in sh-scramble- and PPARγ shRNA-treated mice. n = 5, 6. (C) WGA staining was performed to quantify muscle fiber CSA from sh-scramble- and PPARγ shRNA-treated mice. Scale bar: 20 μm. n = 6 per group. (D) Quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expression in sh-scramble- and PPARγ shRNA-treated mice. n = 5, 6. (E) TUNEL staining was performed to quantify apoptosis from sh-scramble- and PPARγ shRNA-treated mice. Scale bar: 50 μm. n = 4 per group. (F) Western blot analysis for the ratio Bax/Bcl2 and Cleaved-caspase3/Caspase3 from sh-scramble- and PPARγ shRNA-treated mice. n = 3 per group. (G) Western blot analysis for LC3II, P62 and ubiquitin protein expression in sh-scramble- and PPARγ shRNA-treated mice. n = 3 per group. (H) Quantitative real-time PCR analysis the ratio of mtDNA/genomicDNA in sh-scramble- and PPARγ shRNA-treated mice. n = 5 per group. (I) Quantitative real-time PCR analysis of miR-29b expression in sh-scramble- and PPARγ shRNA-treated mice. n…

Figure 8. Overexpression of PPARγ ameliorates AngII-induced muscle atrophy in vivo (A) Quantitative real-time PCR analysis of PPARγ expression in control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. n = 4, 5, 5, 5. (B) Gastrocnemius weight from control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. n = 6. Scale bar is 1cm. (C) The grip strength of right hindlimb from control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. n = 5, 6, 5, 6. (D) WGA staining was performed to quantify muscle fiber CSA from control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. Scale bar: 20 μm. n = 3–4 per group. (E) Quantitative real-time PCR analysis of Atrogin-1 and Murf-1 expressions in control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. n = 5–6 per group. (F) Western blot analysis of Atrogin-1 and MuRF-1 expression in control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. n = 3 per group. (G) TUNEL staining was performed to quantify apoptosis from control and AngII mice treated with Fugw or PPARγ overexpression lentivirus. Scale bar: 20 μm. n = 4 per group. (H) Western…