P mtorc1

Eicosapentaenoic Acid (purity, ≥97%), 3-Methyladenine (3-MA, a PI3K inhibitor) and Dorsomorphin (Compound C, an AMPK inhibitor) were purchased from MedChemExpress (NJ, United States). Type II collagenase, TBHP, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, United States). Primary antibodies for cleaved-caspase 3 (C-C3), Beclin-1, LC3B, p-mTORC1, mTORC1, BCL-2, AMPK and p-AMPK were procured from Cell Signaling Technologies (Danvers, MA, United States). Antibodies against BAX, BCL-2, Collagen II, MMP13 and P62 were purchased from Abcam (Cambridge, United Kindom). Antibodies against p-PERK, PERK, p-eIF2α, eIF2α, CHOP, ADAMTS5, Aggrecan, and ATG5 were purchased from ABclonal (WH, CHINA). Antibodies against ATF4, GRP78, Collagen II, and β-actin were purchased from Proteintech (NJ, China). Horseradish peroxidase-labeled secondary antibodies, Alexa Fluor^® 488-labeled goat anti-rabbit IgG (H + L) secondary antibody, and Alexa Fluor^® 594-labeled goat anti-mouse IgG (H + L) secondary antibody were purchased from Abcam. Further, 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (SH, CHINA). The reagents for cell culture were obtained from Gibco (Grand Island, NY, United States).

Ang II was purchased from ENZO (ALX-151-039-M025); collagenase and trypsin were purchased from Gibco (Grand Island, NY, United States); the BCA protein assay kit was bought from Pierce (Rockford, United States); and 2,7-dichlorofluorescindiacetate (DCFH-DA) was obtained from the Bioengineering Institute (Nanjing, China). The following primary antibodies were obtained from Cell Signaling Technology (CST, United States): glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118), p-mTORC1 (#2971), T-mTORC1 (#2983), α-actinin (#69758S), P-p70 S6 kinase (Thr389) (#9234P), T-p70 S6 kinase (#2708), P-JNK (T183/Y185) (#4668P),T-JNK (#9258), P-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370P), T-ERK (#4695), P-p38 (#4511P), p38 MAPK (#9212P), T-TAK1 (#5206), P-TAK1 (#4508), T-AKT (#4691), P-AKT (#4060), acetyl-CoA carboxylase antibody (#3676), and P-acetyl-CoA carboxylase antibody (#3661S). ABCAM provided the following primary antibodies: Anti-AMPKα2 (ab3760), p-AMPKα2 (S491, ab109402), anti-SOD1 (ab16831), anti-SOD2 (ab38155), Nrf2 (ab15323), 4-hydroxynonenal (ab46545), sarcomeric α-actinin (ab68167), heme oxygenase1 (ab-13243), and NOX2/gp91phox (ab129068). The Sesn2 antibody was acquired from Proteintech (no. 10795-1-AP). Antibodies were used at 1:1,000 dilutions for Western blotting. The secondary antibodies were obtained from LI-COR Biosciences (Lincoln, United States).

Frozen gastrocnemius muscle tissue was ground into small pieces in liquid nitrogen and lysed in a lysis buffer containing cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ (Roche Diagnostics, Indianapolis, IN, USA), and then centrifuged. A Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to measure the protein concentration and same concentration of protein of each group was subjected to SDS-PAGE. After transferring, membranes were blocked by 5% skim milk and incubated with a primary antibody overnight. The primary antibodies used for Western blot were p-Akt, Akt, p-mTORc1, mTORc1, p-S6K1, S6K1, p-4E-BP1, 4E-BP1, p-FoxO3a, and FoxO3a (Cell signaling, MA, USA), MuRF1 and Atrogin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (GeneTex, Irvine, CA, USA). The next day, the membranes were incubated with the corresponding secondary antibodies for visualizing the protein bands using LAS3000 luminescent image analyzer (Fuji Film, Tokyo, Japan). β-actin was used as a loading control and Image J software (National Institute of Health, Bethesda, MD, USA) was used for quantification.