Activity of repurchased compounds on cell death induced by DUX4 or other cytotoxic pathways. (A) Viability of DUX4-expressing C2C12 cells exposed to various concentrations of compounds, from 0.01 μM to 7.44 μM. Compounds are arranged in order of greatest viability at any concentration. Four Prestwich compounds are also shown: α-tocopherol, vitamin K2, ethoxyquin, and ethopropazine HCl. B-G: Viability of C2C12 cells exposed to various cell death-inducing compounds in the presence of 5 μM compounds 1 to 52 (the active compounds of the 54 that were purchased). The Y axis represents viability (ATP content). The first point in each series represents untreated cells, the second represents cells treated with toxic agent alone, and the remaining points represent cells treated with toxic agent plus inhibitory compounds, in order from 1 to 52 (n = 3), error bars = SEM. The dashed red line represents 3 standard deviations above the control sample without compound. (B) Protection from ABT-263. (C) Protection from Staurosporine. (D) Protection from Etoposide. (E) Protection from Ionomycin. (F) Protection from tBHP. (G) Protection from Tunicamycin. (H) Viability of 3T3 cells exposed to tBHP in the presence of compounds 1 to 52. (I) Protection from tBHP in C2C12 cells vs. 3T3 cells. R = Spearman’s correlation coefficient. The strong correlation indicates that compounds tend to protect equally well in both cell types. (J) Activity of 52 repurchased compounds on cell death induced by DUX4 in 293 T cells. Viability of 293T cells transfected with vector control (EV), DUX4 plus carrier (DMSO) alone, or DUX4 and treated with of 5 μM compounds 1 to 52 (n = 6). The Y axis represents improvement in viability over DUX4 transfection alone. Red dots represent compounds that gave a statistically significant (P <0.05) improvement in viability over DUX4 treatment with carrier alone.