Sc 12732

The cells were fixed with 4% paraformaldehyde/PBS for 10 min at room temperature. After washing, fixed cells were treated with 0.1% Triton X-100/PBS for 10 min for permeabilization, blocked for 15 min with 6% skim milk (Megmilk Snow Brand)/PBS for anti-Myf5 antibody, or 5% goat serum in 2% BSA/PBS for the other antibodies. The cells were then incubated with anti-Pax3 (1:80; Developmental Studies Hybridoma Bank), anti-Pax7 (1:80, sc-81648; Santa Cruz), anti-MyoD (1:400, sc-32758; Santa Cruz), anti-myogenin (1:1000, sc-12732; Santa Cruz), or anti-MyHC antibody (1:500, MAB4470, Clone: MF20; R&D Systems) in 2% BSA/PBS, or anti-Myf5 antibody (1:200, sc-302; Santa Cruz) in 6% skim milk/PBS at 4 °C overnight, followed by incubation with Alexa Fluor 488- or Alexa Fluor 647-labeled secondary antibodies (1:1000; Thermo Fisher Scientific) in 2% BSA/PBS. After several washings, nuclei were stained with DAPI (Dojindo). Immunofluorescent staining images were evaluated by fluorescence microscopy (Olympus).

The HSkMSCs were seeded at 2 × 10⁴ cells/well into a 24-well plate on coverslips and were grown in a growth medium for 24 h; then, the culture medium was replaced with a fresh medium containing 0 and 25 μg/mL of exosomes. After 9 days, the cells were washed twice using cold PBS, fixed with 4% paraformaldehyde/PBS for 1 h at 4 °C, washed three times with PBS, and permeabilized using 0.3% Triton X-100 for 20 min at room temperature. Later, cells were blocked with 3% BSA/PBS for 1 h. The cells were incubated overnight at 4 °C with anti-myogenin (MYOG) (sc-12732, Santa Cruz, CA, USA) or anti-myod (MYOD) (sc-377460, Santa Cruz, USA) and were then incubated with anti-mouse IgG–FITC secondary antibody (Santa Cruz). The cells were counterstained using 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 1 min and were then observed with a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA).

CTX and dihydroethidium (DHE) were obtained from MedChemExpress LLC (HY-P1902A, Princeton, NJ, USA) and Sigma (D7008, St. Louis, MO, USA), respectively. Wheat germ agglutinin (WGA) was purchased from Biotium Inc. (29022, Fremont, CA, USA). The ADMA ELISA kit and TUNEL staining kit were purchased from Bio-Techne Co., Ltd. (NBP2–66728, Minneapolis, MN, USA) and Beyotime Institute of Biotechnology (C1090, Shanghai, China), respectively. Lactate dehydrogenase (LDH) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (A020-2-2, Nanjing, China). Antibodies against DDAH1, peroxiredoxin 3 (PRDX3), PRDX5 and β-tubulin were purchased from Signalway Antibody LLC (37368, 38567, 38828, 48659, Greenbelt, MD, USA). Antibodies against Bcl-2, Bax, DDAH2, Interleukin-6 (IL-6), M-cadherin and TNFα were purchased from Abcam (ab194583, ab182733, ab184166, ab259341, ab129078 and ab183218, Cambridge, UK). Antibodies against F4/80 and iNOS were purchased from Wuhan Sanying Biology Technology Company (28463-1-AP and 22226-1-AP, Wuhan, China). Antibodies against MyoD and myogenin were obtained from Santa Cruz Biotechnology, Inc (sc-32758 and sc-12732, Dallas, TX, UA).

After the muscle samples were pulverized using a mortar and pestle in liquid nitrogen or the cells were washed with PBS, RNAiso (108‐95‐2, Takara) was added, and total RNA was isolated with Qiagen RNAeasy Mini Kits (1062832, Qiagen) per the manufacturer's guidelines. The PrimeScript™ RT reagent kit with a gDNA eraser was used to produce cDNA. PowerUp™ SYBR™ green master mix (A25742, Thermo Fisher Scientific) was added to detect the expression of related genes, and the gene‐specific primers are listed in Table S3 and Table S4. Quantitative real‐time PCRs were performed with an ABI QuantStudio 3 machine (Thermo Fisher Scientific).
RIPA lysis solution with 1% PMSF was used to extract total protein. Protein quantification was performed using the BCA method. Gel electrophoresis and membrane transfer were performed according to standard processes. After nonspecific antigen blocking with 5% BSA, primary antibodies including Tent5a (1:200, A12765, ABclonal), MHC (1:50, MF20, DSHB), MHC I (1:50, BA‐D5, DSHB), myogenin (1:500, sc‐12732, Santa Cruz), and Gapdh (1:5000) were used to incubate the polyvinylidene fluoride transfer membrane overnight. Images were captured with the Chemidoctm Imaging System. The relative expression of Tent5a, MHC, and MHC I was calculated according to the gray values using ImageJ software (Version 1.52 V).

C2C12 myoblast differentiation was determined by immunofluorescence staining. Primary monoclonal and polyclonal antibodies against MEF2C (#5030 s, 1:200, CST), MyoG (sc-12732, 1:150, Santa Cruz), MyHC (sc-20641, 1:150, Santa Cruz), MyHC-I (NOQ, 1:200, ab234431) and MyHC-II (My32, 1:200, ab51263) were added to each well in every group and incubated for 12 h at 4 °C. The cells were washed with PBS 3 times for 15 min and incubated with appropriate fluorescent dye-labeled secondary antibodies (Jackson Lab, 1:500, USA) at 25 °C for 2 h. The nuclei were stained with DAPI (Molecular Probes). The images for each group were photographed under a Nikon 80i fluorescence microscope [16 (link)].