To visualize DUX4-induced cell death, ~1.2 × 105 to 1.25 × 105 cells were plated on each well of a 12-well plate (353043, Falcon). The following day, the medium was replaced with medium containing the indicated compounds. After the indicated exposure time, cultures were visualized by phase-contrast microscopy using a Nikon Eclipse TS100 microscope.

To visualize caspase-3/7 activation, 5 × 104 cells per well were plated on four-chambered slides in 0.6 ml of media with or without DOX and either 30 μM InSolution Caspase-3 Inhibitor II (Z-DEVD-FMK; 264156, EMD/Millipore) or an equal volume of DMSO vehicle. After 23 hours, cells were stained by adding either two drops of ReadyProbes CellEvent Caspase-3/7 Green reagent (R37111, Invitrogen) per well or Hoechst dye (1 μg/ml), and the cultures were placed in the incubator for 1 hour. Cells were then imaged on a Nikon Eclipse TS100 microscope.

To quantitate apoptosis and cell death, 1.75 × 105 cells per well were plated on six-well plates. The next day, medium was replaced with medium containing the indicated compounds. After 24 hours, the medium was transferred to a 15-ml centrifuge tube. Wells were rinsed with PBS, which was transferred to the same tubes. Cells were then trypsinized with TrypLE Express (12605-010, Gibco), and reactions were stopped with an equal volume of media and added to the tubes. Wells were rinsed with PBS, which was also added to the tubes. The tubes were spun at ~400g for 5 min. The medium was removed, and the cells were suspended in PBS and then added to 5-ml flow cytometry tubes by passing through a 40-μm nylon strainer (352340, Falcon). Cells were then stained using the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (C10427, Invitrogen) according to the manufacturer’s instructions, with minor adjustments for volume. Flow cytometry was performed using a BD FACSCalibur instrument, and the data were analyzed using FlowJo software.

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