Myogenin

Mouse skeletal muscle-derived C2C12 myoblasts (GNM26) were purchased from the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) was purchased from Clark Bioscience (Richmond, VA, USA). Horse serum was purchased from GIBCO (Life Technologies, Carlsbad, CA, USA). DEX was purchased from Sigma (St. Louis, MO, USA). The lactic dehydrogenase (LDH) Biochemical Analysis Kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). Fluo-3/AM and the BCA Protein Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). Protease/phosphatase inhibitor cocktails were purchased from Roche (Basel, Switzerland). Primary antibodies against myogenin, MuRF 1, atrogin-1, phosphorylated AMPK (p-AMPK), AMPK, phosphorylated PI3K (p-PI3K), PI3K, phosphorylated Akt (p-Akt), Akt, GLUT4, and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). p-AS160 was obtained from OmnimABs (Alhambra, CA, USA). Chemiluminescence reagents were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The C2C12 mouse myogenic progenitor cells line was procured from the American Type Culture Collection [ATCC, Rockwille, MD, USA]. Dulbecco’s modified Eagle’s medium [DMEM-30-2002] and fetal bovine serum (FBS-30-2020) were procured from ATCC [Rockwille, MD, USA]. Kits for mRNA extraction, iScript cDNA synthesis and qPCR were purchased from Bio-Rad [Biorad- California, USA]. FN, Vitamin C and β-glycerophosphate were obtained from Sigma Aldrich (St. Louis, MO, USA). SB203580 inhibitor was provided by Cell Signaling Technology (Danvers, MA, USA). BMP-4 BMP-2, MyoD, Myogenin, α-tubulin, myosin heavy chain (MHC), MHCIIa, MHCIIc, JAK/pJAK1(Tyr1034/1035), JAK2/pJAK2(Tyr1008), STAT1/pSTAT1(Ser727), STAT2/pSTAT2(Tyr690), Smad/Smad1(ser463/465)/5(ser 463/465)/9(Ser465/467), p38MAPK/pp38MAPK (Thr180/Tyr182), ERKs/ERK(Thr202/Tyr204) and Akt/pAkt (Ser473) were acquired from Cell Signaling Technology (Danvers, MA, USA), Abcam (Cambridge,UK) and Santa Cruz Biotechnology (Dallas,Texas, USA).

C2C12 cells were seeded at 2 × 10⁵ cells/cm² into a 6 cm dish, and cell lysates were collected after VV stimulation from days 1 to 3. RIPA Lysis and Extraction Buffer (89900, Thermo Fisher Scientific Inc., Waltham, MA, USA) containing protease and phosphatase inhibitor (1:100; A32961, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to extract the cell lysate. Western blot analysis was performed with primary antibodies against stathmin (1:1000), decorin (1:1000), Col-I (1:500), FGF7 (1:1000), TGFBr1 (1:500) and PAK3 (1:500) (Abcam Inc., Cambridge, MA, USA), pAKT (1:500), AKT (1:2000), MyoD (1:1000) and myogenin (1:500) (Cell Signaling Inc., Danvers, MA, USA), β-actin (1:10,000) (Arigo Biolaboratories Corp., TE Huissen, Netherlands), which were incubated with the membranes at 4 °C overnight. After being washed 3 times for 15 min each, the membranes were incubated with an HRP-conjugated goat antibody anti-mouse or anti-rabbit IgG secondary antibody (Arigo Biolaboratories Corp., TE Huissen, Netherlands) for 1 h at RT. The proteins were detected using an ECL substrate kit (Abcam Inc., Cambridge, MA, USA). Band intensity was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Intensity data were normalized to GAPDH and indicated controls for the target protein fold changes calculation.