F 10 ham

The panel was composed of commercial and in-house-generated cell lines from patients of soft tissue sarcomas. For the generation of new cell lines, sterile fragments from resected tumors were minced in culture medium and then disaggregated by 1–2 h incubation in collagenase (100 U/ml) at 37°C. After 24 h, the medium was changed to F-10 Ham (Gibco) supplemented with 1% Ultroser G (Biosepra). The cell lines generated were cultured in F-10 Ham supplemented with 1% Ultroser G. A673 cells were cultured in RPMI (Sigma) and SW872 in Leibovitz L-15 (Sigma). All media were supplemented with 10% FBS, fungizone, and penicillin/streptomycin. Once the cells became confluent, adherent cells were removed by trypsin treatment and seeded at 1/2 or 1/3 ratio with medium. Throughout the establishment of these cell lines, their phenotypic features were followed. Additionally, the cell lines were routinely checked for mycoplasma contamination (INVIVOGEN). All cell lines used were established immortal tumor cell lines.
For the newly created human cell lines from resected tumor tissue, approval from local ethics committee at Hospital Universitario Virgen del Rocio (Comite etico de investigacion Hospital Universitario Virgen Del Rocío) was obtained (PI2012-0085) and informed consent was obtained from patients.

Mice testis-derived Leydig cell culture I-10 cells (RIKEN BRC, Tsukuba, Japan) and MA-10 cells (ATCC, Manassas, VA, USA) were maintained in a nutrient mixture F-10 Ham (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), 50 U/mL penicillin, and 50  µg/mL streptomycin (Gibco, Thermo Fisher Science, Tokyo, Japan). Cells were maintained at 37 °C and 5% CO₂/95% air, as controlled by the incubator. The maximum levels of biotin and additive agents were determined at concentrations that did not affect cell viability, using the Premix WST-1 Cell Proliferation Assay System (Takara Bio, Shiga, Japan).

Cell preparations were collected from the entire hindlimb and forelimb of E18.5 embryos. The skin was removed, and the limbs were minced and digested at 37 °C with 1.15 mg/ml of collagenase type IV (Sigma) and 0.4 mg/ml dispase (Life Technologies) for 20 min. Tissue was filtered through 70 and 40 μm cell strainer and blood cells were lysated by ammonium chloride (Stem Cell Technologies). Cells were collected by centrifugation and resuspended in nutrient mixture F-10 Ham (Sigma) supplemented with 20% fetal bovine serum (FBS) (Hyclone), 1% penicillin–streptomycin, 1% glutamine (Gibco), 0.1% gentamicin (Sigma), and 5 ng/ml basic fibroblast growth factor (Preprotech). Then, cells were plated on collagen-coated dishes, after 30 min pre-plating in uncoated dishes (three incubations). Primary myoblasts were grown for 2 days and then differentiated in Dulbecco’s modified Eagle's medium (DMEM; EuroClone) supplemented with 5% donor horse serum (EuroClone) for 48 h. Cells were collected and RNA extraction was performed using ReliaPrep RNA cell miniprep system (Promega).