dsDNA breaks were measured using the EMD/Millipore H2A.X Phosphorylation Assay Kit (17-344), according to the manufacturer’s instructions with minor modifications for volume. Briefly, 1.75 × 105 MB135-DUX4i myoblasts were plated per well on six-well plates. The following day, the test compounds were added to the indicated concentrations. After 24 hours, the medium was transferred to 15-ml centrifuge tubes. Wells were rinsed with PBS, which was added to the centrifuge tubes. The cells were then trypsinized with TrypLE Express, and reactions were stopped with an equal volume of media and added to the tubes. Wells were washed with PBS, which was also added to the tubes. The tubes were then spun down at ~750g for 5 min and were washed three times with PBS. Cells were resuspended in 25 μl of fixation buffer and left on ice for 20 min. Fixed cells were spun down at ~10,000g and washed twice in PBS and then resuspended in 25 μl of permeablization buffer. Fluorescein isothiocyanate–conjugated antibody (1.75 μl) was then added, mixed, and left on ice for 20 min. Cells were then diluted with 100 μl of wash buffer, spun down, washed with Hanks’ balanced salt solution (HBSS; 21-022-CV, Corning) and then resuspended in HBSS and transferred into a flow cytometry tube by passing through a 40-μm nylon strainer. Flow cytometry was then performed using a BD LSR II flow cytometer, and the data was analyzed using FlowJo software.

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