DENR/MCTS1 knockdown selectively suppresses RAN translation. (A) Western blot showing efficiency of DENR KD in HEK293 cells 48 h post transfection with increasing concentrations of DENR siRNA. GAPDH was used as a loading control. (B) Western blots showing effects of DENR KD on expression of AUG-NLuc and GA70-NLuc reporters expressed in HEK293 cells, 48 h post KD and 24 h post reporter transfection. NLuc reporters were probed with an antibody targeting their c-terminal FLAG tag. GAPDH was used as a loading control. (C–E) NLuc expression of indicated reporters expressed from DNA plasmids transfected into HEK293 cells 24 h post transfection with non-targeting or DENR targeting siRNAs. NLuc levels are expressed relative to levels in cells transfected with the non-targeting siRNA, n = 9. (F) NLuc expression of indicated reporters expressed from DNA plasmids in HEK293 cells 24 h post transfection with non-targeting or MCTS1 targeting siRNAs. NLuc levels are expressed relative to levels in cells transfected with the non-targeting siRNA, n = 9. All graphs represent mean with error bars ± standard deviation. ***P < 0.001, ****P < 0.0001, Two-way analysis of variance with Sidak’s multiple comparison test.