Figure 4 from The XPO1 Inhibitor Eltanexor Modulates the Wnt/β-Catenin Signaling Pathway to Reduce Colorectal Cancer Tumorigenesis

Eltanexor increases FoxO3a nuclear localization to modulate Wnt/β-catenin signaling. A–C, HCT116, RKO, and DLD1 cells (all harboring different Wnt signaling phenotypes) were transfected with a TOPFlash reporter plasmid. Cells were subsequently treated with DMSO or eltanexor. RKO and HCT116 cells were treated with 200 nmol/L eltanexor, and DLD1 cells were treated with 400 nmol/L eltanexor for 24 hours. Luciferase activity/μg of protein was normalized to DMSO-treated luciferase expression for each respective cell line ± SEM. A Student t test was used to statistically compare luciferase activity in the control-treated and eltanexor-treated groups. D, HCT116 cells were treated for 48 hours with either 200 nmol/L eltanexor or DMSO. Subsequently, the cells were subject to immunofluorescent staining for FoxO3a (green). The nucleus was stained with DAPI (blue). E, The mean FoxO3a nuclear fluorescence for cells treated with DMSO (control) or 200 nmol/L eltanexor for 48 hours. The graph depicts the mean of three independent experiments ± SEM. At least 40 cells were analyzed for each independent experiment. The scale bars represent 50 μm. A Student t test was used to compare the mean fluorescence in the control-treated and eltanexor-treated groups. F and G, In HCT116 cells, FoxO3a was knocked down by siRNA. After knockdown, the cells were transfected with a TOPFlash reporter plasmid. The FoxO3a knockdown–transfected and TOPFlash-transfected cells were subject to immunoblotting to confirm FoxO3a knockdown. Actin was used as the loading control. The graph depicts the quantified densitometry of the Western blot bands. The values are normalized to actin and relative to control siRNA treated. The graph depicts the mean of three independent experiments ± SEM. A Student t test was used to statistically compare FoxO3a expression in the control siRNA–treated and FoxO3a siRNA–treated groups. H, The relative luciferase activity/μg of protein values was normalized to control siRNA. A Student t test was used to statistically compare luciferase activity in the control-treated and eltanexor-treated groups. The graph depicts the mean of three independent experiments ± SEM. A Student t test was used to statistically compare the control siRNA–treated and FoxO3a siRNA–treated groups. (*, P ≤ 0.05; **, P ≤ 0.01.)