Skeletal muscle media

HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke Cell Culture Facility and were maintained in DMEM supplemented with 10% fetal bovine calf serum and 1% penicillin/streptomycin. Immortalized myoblasts^{70 (link)} from a DMD patient harboring a deletion of exons 48–50 (Δ48–50) in the dystrophin gene were maintained in skeletal muscle media (PromoCell) supplemented with 20% fetal bovine calf serum (Sigma), 50 μg/ml fetuin, 10 ng/ml human epidermal growth factor (Sigma), 1 ng/ml human basic fibroblast growth factor (Sigma), 10 μg/ml human insulin (Sigma), 1% GlutaMAX (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). All cell lines were maintained at 37°C and 5% CO₂. HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) with 400 ng of each expression vector according to the manufacturer’s protocol in 24 well plates. Immortalized myoblasts were transfected with 5 micrograms of each expression vector by electroporation using the Gene Pulser XCell (BioRad) with PBS as an electroporation buffer using optimized conditions ^{28 (link)}. Transfection efficiencies were measured by delivering an eGFP expression plasmid (pmaxGFP, Clontech) and using flow cytometry. These efficiencies were routinely ≥ 95% for HEK293T and ≥ 70% for the immortalized myoblasts.