RNAscope specifically detected overexpressed DUX4 mRNA in transfected HEK293 cells. (A) DUX4-transfected cells showed punctate brown staining with RNAscope assay, while controls did not. Decreased brown staining with increased doses of DUX4-targeted microRNAs (mi405) demonstrated specificity. (B–D) DUX4 signal after cotransfection of HEK293 cells with CMV.DUX4 and U6.mi405 plasmids at 1:2, 1:4, and 1:8 ratios. (E) Absence of brown DUX4 signal in untransfected HEK293 cell line. (F) RNAscope negative control stain. (G) Housekeeping gene PPIB was detected in all HEK293 cells and served as a positive control for the assay. (H) Cell viability assay demonstrated that mi405 reduced the levels of overexpressed DUX4 mRNA, and significantly protected cells from death at 1:4 and 1:8 weight ratios (N = 3 independent experiments performed in triplicate); P < 0.0001, ANOVA. 40× objective. Scale bar, 50 microns.
