Figure 6.

Biological activity of CB096 in a HEK293T cellular model of c9ALS/FTD. (a) CB096 selectively inhibits repeat-associated non-ATG (RAN) translation of r(G₄C₂)^exp. Data indicated with blue bars were collected from HEK293T cells cotransfected with plasmids encoding r(G₄C₂)₆₆-No ATG-GFP and ATG-mCherry, while data indicated with purple bars were collected from HEK293T cells cotransfected with plasmids encoding ATG-GFP and ATG-mCherry. Data are reported as the mean ± SD from three independent measurements each with three technical replicates. (b) Effect of CB096 on r(G₄C₂)₆₆ mRNA levels, as measured by RT-qPCR. Data are reported as the mean ± SD from three independent measurements with three technical replicates each. “n.s.” indicates not significant. (c) Effect of CB096 on the levels of toxic dipeptide repeats of poly(GP) upon 24 h of treatment. Poly(GP) levels were measured by an electroluminescent sandwich immunoassay. Data are reported as the mean ± SD and are representative of two independent measurements with two technical replicates each. (d) Rescue of nucleocytoplasmic transport dysfunction by CB096. Representative images of HEK293T cells stably expressing Lentiviral-S-tdTomato cotransfected with plasmid encoding a (G₄C₂)₆₆-No ATG-firefly luciferase (RAN translation), SV40-Renilla (canonical translation), and ATG-GFP (for distinguishing transfected from non-transfected cells in imaging studies). Cells were treated with vehicle, KPT-350 (KPT at 10 μM; a selective inhibitor of Exportin-1-mediated nuclear export), or CB096 (25 μM). (e) Quantification of the percentage of tdTomato localized to the nucleus or cytoplasm (n = 3 biological samples; 200 cells evaluated per biological sample). (f) Effect of CB096 on r(G₄C₂)₆₆-induced or chemically induced stress granules. For the former, HEK293T cells stably expressing Lentiviral-S-td-Tomato were transfected with (G₄C₂)₆₆-No ATG-GFP and treated with CB096 or vehicle (25 μM). For chemically induced stress granules, HEK293T cells stably expressing Lentiviral-S-td-Tomato were transfected with ATG-GFP and treated with 0.5 mM sodium arsenite (NaAsO₂). Stress granules were visualized by Ataxin-2 (ATXN2) staining and imaged by confocal microscopy. (g) Quantification of average number of stress granules per nuclei upon treatment with vehicle or CB096 (n = 3 biological samples; 200 cells counted per biological sample). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined by one-way ANOVA. Data are reported as mean ± SD.