The inducible DUX4 myoblast model MB135-DUX4i and its parental cells (33) were provided by S. Tapscott. The line was routinely tested for mycoplasma contamination by the provider. We confirmed identity by ability to induce DUX4 expression and ability to form myotubes and confirmed that it is not present in the Register of Misidentified Cell Lines. 15ABicCT, 15BBicCT, and 15VBicCT immortalized human myoblasts were provided by W. E. Wright (50). We confirmed their identity by confirming their ability to form myotubes and by the induction of DUX4 target genes in appropriate cell lines after formation of myotubes, and they were not present in the Register of Misidentified Cell Lines. We confirmed that all five lines are free of mycoplasma contamination. All cells were grown on vessels precoated with 0.1% gelatin (G939, Sigma-Aldrich). MB135-DUX4i and parental myoblasts were propagated in 20% characterized fetal bovine serum (FBS; SH30071.03, Hyclone), dexamethasome (51 ng/ml; D2915, Sigma-Aldrich), fibroblast growth factor-basic (bFGF, 10 ng/ml; GF003AF-MG, EMD Millipore), and 1× antibiotics/antimycotics (30-004-CI, Corning) in Ham’s F10 medium (10-070-CV, Corning). 4MU (M1381, Sigma-Aldrich) was diluted in the appropriate culture media from a 100 mM stock in DMSO or 100% ethanol. DUX4 was induced by adding doxycycline (DOX) hyclate (D9891, Sigma-Aldrich) to 2 μg/ml from a stock (2 mg/ml) in water. H2O2 (216763, Sigma-Aldrich), βME in phosphate-buffered saline (PBS; 2020-05-30, Gibco/Life Technologies), and etoposide were added to the media at the concentrations indicated. These immortalized, patient-derived myoblasts were grown in medium consisting of 15% characterized FBS, zinc sulfate (0.3 μg/ml; Z0251, Sigma-Aldrich), vitamin B12 (1.4 μg/ml; V2876, Sigma-Aldrich), dexamethasome (3.5 to 54 ng/ml; D2915, Sigma-Aldrich), human hepatocyte growth factor (2.5 ng/ml; 130-093-872, Miltenyi Biotec), bFGF (10 ng/ml), 1× antibiotics/antimycotics (30-004-CI, Corning), and 20 mM Hepes (25-060-Cl, Corning) in medium X [4:1 mixture of Dulbecco’s modified Eagle’s medium:Medium 199 (10-013-CV and 10-060-CV, Corning)].

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