Ccl 136tm

RD (Human rhabdomyosarcoma; ATCC^®, CCL-136^TM), HEK293T, HeLa and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 100 unit/ml penicillin, 10 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mM L-glutamine and 10% fetal bovine serum (HyClone) at 37°C with 5% CO₂. The EV-A71 genotype C strain (4643/Taiwan/1998), provided by Dr. Shin-Ru Shih (Chang-Gung University, Taiwan), was amplified in RD cells. All experiments using infectious viruses were carried out in biosafety level 2 laboratory, following the guidelines of the Center of Environmental Protection and Safety and Health (National Yang-Ming University, Taiwan).

In vitro models of ERMS were used in this study. Specifically, the RD cell line was purchased from ATCC (CCL-136^TM), whilst the JR1 cell line was kindly provided by Dr. Rota R (Bambino Gesù Children’s Hospital, Rome, Italy). All the cell lines were maintained in complete medium, i.e., high-glucose DMEM (DMEM-HG, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% foetal bovine serum (FBS) (Corning, New York, NY, USA), 2 mM l-glutamine (Corning), 100 IU/mL penicillin and 100 μg/mL streptomycin (Corning) and grown at 37 °C in 5% CO₂.
SB203580, a p38-specific inhibitor, was purchased from Selleckchem (Suffolk, UK) as lyophilized powders and were reconstituted in DMSO to a final concentration of 5 mM. SB203580 was added to the cultured medium 24 h after specific transfection to a final concentration of 5 μM.