Ultroser g

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.

Total muscle explant-derived cells were purified by Magnetic-activated cell sorting (Milteny Biotech) using anti-CD56 antibody (Table S1). Myoblast identity was determined by desmin immunostaining (> 98%). The primary and immortalized myoblasts were grown, respectively, in DMEM with high glucose and l-glutamine (Lonza), 10% fetal bovine serum (FBS) (Invitrogen), 1% Ultroser G (Pall BioSepra, Cergy-St-Christophe, France), and gentamicin (50 μg/ml, Sigma-Aldrich) or DMEM high glucose supplemented with 16.5% medium 199 (Lonza), 15% FBS, Ultroser G, HEPES 1 M (Sigma-Aldrich), zinc sulfate (Sigma ®-Aldrich, vitamin B12 (Sigma-Aldrich), and penicillin/streptomycin (pen/strep) at 37 °C under atmosphere with 5% CO₂. For myogenic differentiation, cells were cultured on Matrigel-coated culture dishes and a differentiation medium was added after cells reached 100% confluence. This medium was composed of DMEM/gentamicin (50 μg/ml) with 2% FBS for primary cells and DMEM high glucose, medium 199 supplemented with 0.5% insulin, 1% apo-transferrin (Sigma-Aldrich), 2% HEPES 1 M and pen/strep for immortalized cells. HEK293 were grown in DMEM high glucose-10% FBS and pen/strep. Transfection of primary cells was previously reported [3 ]. The KLF15 expression vector was a generous gift of Prof. Yegor Vassetzky [18 (link)].