Wnt pathway activation by extracellular Wnt3a as reflected in nuclear β-catenin content and level of Wnt throughput in EAhy926 endothelial cells. (A) Control cells were not treated with Wnt. (B) Cell treatment with Wnt3a increased their cytoplasmic and nuclear β-catenin content. Cells were analyzed by immunofluorescence with primary antibodies against β-catenin, biotinylated secondary antibody, and subsequently TSA Fluorescein System. (C) A typical control cell with β-catenin staining (green fluorescence) and nuclear staining (blue fluorescence, DAPI). (D) A typical Wnt3a-treated cell. After treatment of cells with Wnt3a conditioned media (CM) β-catenin staining co-localized with nuclei. (E) Ratio of nuclear to cytoplasmic β-catenin in the EAhy926 cells. Wnt3A CM increased the nuclear/cytoplasmic ratio (**p < 0.0001). (F) Wnt throughput was measured by the SuperTOPflash reporter construct in EAhy926 cells. Wnt3a CM increased Wnt throughput (**p = 0.0038). No activation of the same reporter with mutated Lef/Tcf binding site (SuperFOPflash) indicated specificity of the Wnt3a-dependent cell response. (G) Wnt throughput as measured by the SuperTOPflash reporter construct in EAhy926 cells. Exposure to Wnt3a by side-by-side co-culture also increased Wnt throughput (**p = 0.0034). The SuperFOPflash reporter was used to check specificity of the response.