Figure 3.

WNT-3A induces TCF/LEF transcriptional activity in the absence of functional heterotrimeric G protein signaling. A, HEK293 and ΔG7 HEK293 cells were transfected with M50 Super 8xTOPFlash and Renilla luciferase. After transfection, cells were serum-starved either in the absence or presence of PTX (100 ng/ml) for 4 h. Subsequently, cells were stimulated with increasing concentrations of purified, recombinant WNT-3A for 20 h. Luciferase data were normalized to the maximal response of parental HEK293 cells to 2000 ng/ml WNT-3A in the absence of PTX. Three independent experiments were done in duplicates. Data were fit to a four-parameter nonlinear regression using GraphPad Prism 6. Error bars, S.E. B, the table summarizes EC₅₀ values and maximal effects of the TOPFlash signal evoked by WNT-3A under the different experimental conditions. C, TOPFlash activity was quantified in ΔG7 HEK293 cells, which were transfected with WT or constitutively active QL mutants of Gα proteins representing the four different families of G proteins (G_s, G_i1, G_q, and G₁₂). Data are presented as the mean of four independent experiments performed in duplicates. Error bars, S.D. Data were normalized to pcDNA-transfected cells (red dotted line). Results were analyzed with one-way ANOVA and Tukey's multiple-comparison post hoc test. Significance levels are indicated as follows: *, p < 0.05, comparison with pcDNA.