Secondary screens. (A) Effects of compounds on cell proliferation. (B) Effects of compounds on a dox-inducible luciferase transgene. Compounds inhibiting luciferase expression are predicted to also inhibit DUX4 expression and thus give a false positive increase in viability. (C) Protection of an independent cell line (i3T3-DUX4) from cell death induced by DUX4. (D) Screening tree. At left is the graphed the maximum viability at any dose of each compound in the repeat of the primary screen. Green circles indicate compounds considered to have passed this screen (that is, repeated the primary screen by protecting cells against DUX4), and the activity of these compounds is indicated in the following panel. The middle panel shows the activity of the selected compounds on the first two secondary screens: effect on growth rate shown on the y-axis, and inhibition of luciferase (minimum luciferase expression at any concentration of compound) on the x-axis. All compounds are plotted, those with activity in the previous assay are shown as green circles, those that failed the previous assay are shown as gray circles. The box indicates cutoffs set for growth rate and dox-inducible luciferase assays. The third panel shows the activity of selected compounds (that is, those within the box, which did not alter growth rate or affect dox-inducible luciferase expression levels) against DUX4-expressing 3 T3 cells on the y-axis vs. the repeat of the primary assay (same data as the far left panel) on the x-axis. Compounds indicated in green protect against DUX4 independent of cell type.