Fig. 3.

Ouabain inhibits β-catenin signaling, macropinocytosis, and cell proliferation. (A) β-catenin transcriptional activity stimulated by Wnt3a was strongly inhibited by Ouabain in HEK293BR cells. (B) Untreated HEK293BR (BAR/Renilla) cells stained with total β-catenin antibody. (C) HEK293BR (BAR/Renilla) cells treated with Ouabain showed no changes in β-catenin levels. (D) LiCl (40 mM) activates the β-catenin transcriptional response as shown in the BAR-luciferase assay. This effect was blocked with Ouabain (1 µM) treatment. (E) The increase of β-catenin levels due to LiCl, which increases macropinocytosis (Albrecht et al., 2020), was blocked by Ouabain. (F) Quantification of the β-catenin levels from B and E. (G) Quantification of the macropinocytosis uptake of TMR-dextran 70 kDa into SW480 cells and its reduction by Ouabain treatment. (H) TMR-dextran 1 (1 mg/ml) uptake into SW480 cells after 1 h of incubation. (I) TMR-dextran uptake after 1 h of incubation is reduced with Ouabain (200 nM) treatment. (J-K) SW480 cells transfected with membrane-GFP display sustained macropinocytic vesicles (arrowhead and inset) that were rapidly blocked within 30 min of Ouabain treatment (still images from Movie 1). (L) Cell proliferation in SW480 cells is decreased by Ouabain treatment (200 nM), only viable cells were scored. All experiments with cultured cells were biological triplicates. Error bars denote SEM (n≥3) (**P<0.01, ****P<0.0001). Scale bar: 10 μm.