H9 hesc line

DMD patient and control-derived myoblasts were obtained from Dr. Gabsang Lee using a previously published protocol^{69 (link)}. DMD myoblasts include those from a 9-year-old patient with an EX4-43DEL mutation (05169) as well as an 18-year-old patient with a c.5533G>T, p.E1845X mutation (05127). The DMD myoblasts were originally obtained from fibroblasts (Coriell Institute for Medical Research Catalog #GM05169 and #GM05127, respectively) that were reprogrammed into human pluripotent stem cells (hPSC), whereas the control myoblasts were originally obtained from human embryonic stem (hESC). The H9 hESC line was purchased from WiCell (WA09) and is listed on the Human Pluripotent Stem Cell Registry (hPSCReg ID: WAe009-A), where details of informed consent are provided. Details surrounding patient informed consent for the fibroblasts purchased from Coriell #GM05169 and #GM05127 could not be provided due to confidentiality. Both DMD hPSCs and control hESCs were directed towards a skeletal muscle cell lineage and converted into myoblasts using a chemical compound-based approach^{69 (link)}. In addition to the healthy control, an isogenic control was also included. The isogenic control myoblasts are genetically corrected DMD myoblasts whereby the entire dystrophin gene was inserted into the GM05169 DMD fibroblast before its myogenic conversion. Once the myoblasts were shipped to Brock University, they were cultured in growth medium [Dulbecco’s modified Eagle’s medium (DMEM), D6429 Sigma, Oakville, ON, Canada] supplemented with 10% fetal bovine serum (Sigma F1051), 1% penicillin/streptomycin (Sigma P4333), and 2% non-essential amino acids (Sigma M7145) at 37 °C in a humidified atmosphere with 5% CO₂ and 5% O₂ levels^{15 (link)}. After reaching confluence, the cells were trypsinized and pelleted. The pellets were then lysed in RIPA buffer and stored at −80 °C for western blot analysis.