Crl 2061

The rhabdomyosarcoma cell line CRL-2061 and the cell line HEK293 were purchased from ATCC (Manassas, VA, USA). CRL-2061 cells were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) (Gibco by Life Technologies, Grand Island, New York, NY, USA) and 1% antibiotic-antimycotic solution (Nacalai Tesque, Inc.) at 37 °C in a 5% CO₂ humidified incubator. HEK293 cells were cultured in DMEM (High Glucose) (Nacalai Tesque, Inc.) supplemented with 10% FBS and 1% antibiotic-antimycotic solution at 37 °C in a 5% CO₂ humidified incubator.
Human myoblasts that were generated by immortalization of primary cultured human myogenic cells and used in a myoblast proliferation study were a kind gift from Dr. Hashimoto. The myoblasts were grown in DMEM (High Glucose) supplemented with 20% FBS, 2% Ultroser G (Pall Life Sciences, Washington, NY, USA) and 1% antibiotic-antimycotic solution (Nacalai Tesque, Inc.) at 37 °C in a humidified incubator with 5% CO₂.

The HEK293 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco/Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, Moregate Biotech, Bulimba, Australia) and 1% antibiotic-antimycotic reagents (Gibco/Life Technologies). The SHSY-5Y and CRL-2061 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SHSY-5Y cells were cultured in a 1:1 mixture of DMEM and Ham’s F12 (Wako Pure Chemical Industries Ltd., Osaka, Japan), and CRL-2061 cells were cultured in RPMI medium (Gibco/Life Technologies). These were supplemented with 10% FBS (Gibco/Life Technologies) and 1% antibiotic-antimycotic reagents. All cell lines were cultured at 37 °C in a 5% CO₂ humidified incubator. Prior to use, cultured cells were rinsed twice with phosphate-buffered saline (PBS, Sigma–Aldrich Co., St. Louis, MO, USA) and collected using a cell scraper.

The human alveolar RMS (ARMS) cell lines CRL-2061 (PAX3-FKHR translocation positive), the embryonal RMS (ERMS) cell lines RD and TE671 as well as the mouse fibroblast cell lines WNT3A (CRL-2647) and L cells (CRL-2648) were obtained from the American Type Culture Collection (ATCC, Menasses, VA, USA). The translocation negative ARMS cell line FLOH1 was a kind gift from Ewa Koscielniak, Stuttgart, Germany. CRL-2061 and FLOH1 were cultivated in RPMI1640 (Gibco, Carlsbad, CA, USA) with 10% (v/v) FCS (Sigma Aldrich, St. Louis, MO, USA). RD, TE671 and the mouse fibroblast cell lines WNT3A and L cells were maintained in DMEM (Gibco) with 10% (v/v) FCS. The conditioned medium with and without WNT3A was produced as described (ATCC). No antibiotics were added during cultivation. For all experimental settings cell cultures with similar cell densities were used to guarantee comparability of the results. These were for all cultivation conditions around 90%, since cell contacts are required to induce muscle differentiation.
All RMS tumor cells were sequenced by Sanger Sequencing to exclude potential activating β-catenin mutations.