Non essential amino acid solution

Cells were maintained in Dulbecco’s modified Eagle’s medium (Merck, Darmstadt, Germany) containing 10 % fetal bovine serum, 1 % penicillin–streptomycin solution, and 1 % non-essential amino acid solution (Nacalai Tesque, Kyoto, Japan) in an atmosphere of 5 % CO₂.
Cells that expressed GFP, CD90.2, and/or CAR were generated using lentiviral vectors. Briefly, the GFP, mouse CD90.2, and human CAR genes were cloned into the BamHI and EcoRI sites of the FG11BSE lentiviral vector. All vesicular stomatitis virus (VSV)-G pseudotyped lentiviral vectors were produced via the calcium phosphate-mediated transient transfection of 293 T cells. Then 293 T cells were co-transfected with appropriate amounts of vector plasmid, the HIV-1 lentiviral packaging constructs pRSVREV [27 (link)] and pMDLgpRRE [27 (link)], and the VSV-G expression plasmid pHCMVG [28 (link)]. The lentiviral vectors were collected from the culture supernatants 3 days after transfection. Cells were incubated with lentiviral vectors for 3 days, followed by the sorting of transduced cells using a cell sorter (SH800, Sony, Tokyo, Japan). GFP, CD90.2, and CAR expression on the surface of sorted cells was confirmed using flow cytometry. Ad5fiber-expressing 293 (Ad5fiber 293) cells, which we generated previously, were used [29 (link)].

Breast muscles dissected from 14-day-old chick embryos were minced using surgical scissors and digested with HBSS (+) (Nacalai Tesque, Inc., Kyoto, Japan) containing 0.2% collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) for 20 min at 37°C. The cells were collected by centrifugation and resuspended in DMEM (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 15% serum (Fetal Clone III, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), 1× non-essential amino acid solution (Nacalai Tesque, Inc., Kyoto, Japan), and 1× gentamicin/amphotericin solution (Life Technologies, Carlsbad, CA, USA). The cell suspension was passed through a cell strainer to remove tissue debris and then transferred to an uncoated flask to allow attachment of fibroblasts. After 1 h, the unattached cells were transferred to another uncoated flask and this procedure was repeated 2–3 times. The unattached cells were counted and plated onto collagen I-coated 12-well plates at 1×10⁵ cells/well. The cells were incubated in the medium described above at 37°C, with 5 % CO₂ in a humidified chamber until the formation of myotubes. Myotubes were treated with or without 20nM recombinant human/rat/mouse myostatin (PeptroTech Inc., Rocky Hill, NJ, USA) for 2 h, in the absence or presence of serum.

The B16-F0 mouse melanoma cell line (cat. no. JCRB0202) was purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank and maintained in Eagle's Minimum Essential Medium (EMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin and 100 µg/ml streptomycin (both from Nacalai Tesque, Inc.) under mycoplasma-free conditions. Clone M3 (Cloudman S91) melanoma cell line was obtained from European Collection of Authenticated Cell Cultures (ECACC) and cultured in Ham's F10 medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 15% FBS, 2 mM glutamine (Nacalai Tesque, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. 293T and 293FT cells were obtained from Invitrogen; Thermo Fisher Scientific, Inc. HEK-Blue™ TGF-β cells were purchased from InvivoGen. 293T, 293FT and HEK-Blue™ TGF-β cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cultured medium for 293FT cells was also supplemented with 1% non-essential amino acid solution (Nacalai Tesque, Inc.). All cell lines were cultured in a humidified incubator containing 5% CO₂ at 37°C.