Fig. 3 Reduction of SUPT4H1 or SUPT5H in c9ALS fibroblasts inhibits production of C9orf72 variant 3 mRNA, sense and antisense RNA foci, and DPR proteins.

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Fig. 3 Reduction of SUPT4H1 or SUPT5H in c9ALS fibroblasts inhibits production of C9orf72 variant 3 mRNA, sense and antisense RNA foci, and DPR proteins. Reduction of SUPT4H1 or SUPT5H in c9ALS fibroblasts inhibits production of C9orf72 variant 3 mRNA, sense and antisense RNA foci, and DPR proteins. Cultured fibroblasts from three c9ALS patients were treated with a control siRNA (siCtrl), siRNA(s) directed against SUPT4H1 and/or SUPT5H (siSUPT4H1 and siSUPT5H, respectively), or a C9orf72-repeat targeting ASO (C9-ASO) for 10 days. After treatment, mRNA levels of SUPT4H1 and SUPT5H were determined by qPCR (A), and SUPT4H1 and SUPT5H protein expression was examined by immunoblot followed by densitometric quantification (B). (C) The effect of SUPT4H1 and/or SUPT5H depletion on C9orf72 variant 3 mRNA expression and poly(GP) protein levels was examined by qPCR and immunoassay, respectively. (D) RNA FISH with probes for GGGGCC or GGCCCC RNA was used to detect foci containing sense or antisense repeats (red) in the nucleus of cells (blue, Hoechst 33258). The percentage of cells containing foci was then determined. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as assessed by one-way ANOVA followed by Tukey’s post-hoc analysis. Scale bar: 5 μm. Nicholas J. Kramer et al. Science 2016;353:708-712 Copyright © 2016, American Association for the Advancement of Science