Figure 4.

Small molecules inhibit C9RAN translation in all three reading frames. BIX01294 (A), anthralin (B), and protoporphyrin IX (C) were added at increasing concentrations to 30% RRL in vitro translation reactions with AUG-NLuc or GGGGCCx70 reporter mRNAs for all three reading frames, and luminescence was measured relative to vehicle (DMSO) control. D–F, selective inhibition of RAN translation of GGGGCCx70 and CGGx100 NLuc reporter mRNAs, relative to AUG-NLuc control, was confirmed by Western blotting against a C-terminal FLAG tag for the glycine-alanine (GA) and FMRPolyG (+1CGG) products, following incubation with BIX01294, anthralin, or protoporphyrin IX at the indicated doses. GAPDH was used as a loading control. For A–C, all graphs represent mean of n = 3; error bars, S.D. *, p < 0.05, two-way ANOVA with Dunnett's multiple-comparison test. M, mock; GA, glycine-alanine; GP, glycine-proline; GR, glycine-arginine.