Fig. 5

DUX4 and DUX4-target gene expression in response to chemical inhibition of chromatin modifiers. Dose escalations of the HDAC inhibitor RG2833 (a) or the EZH2 inhibitor GSK-126 (c). Cultured myoblasts were treated with the vehicle or the indicated drug at the time differentiation is initiated. Luciferase output from a DUX4-reporter is measured 48 h later, and cell numbers are estimated using the fluorescence signal from CellTiterFluor. Shown is the fold change in luciferase output normalized to cell number when the treatment is compared to vehicle alone. Analyses of all samples were performed in quadruplicate. Reductions in luciferase signal seen at higher drug concentrations (RG2833 25–50 µM and GSK-126 20–50 µM were a consequence of decreased myoblast fusion). Gene expression of DUX4 and its secondary targets CCNA1 and MBD3L2 for RG2833 at 10 µM concentration (b) and GSK-126 at 10 µM concentration (d) are shown. RNA expression was normalized to GAPDH (for DUX4) and RnaseP (for CCNA1 and MBD3L2) as endogenous controls. Analyses of all samples were performed in triplicate. p values were calculated using the Student’s t test to compare vehicle and the treated group with *p ≤ 0.05 as being significant