Figure 3. Wnt5a-Mediated Inhibition of Canonical Signaling Is Not Associated with Wnt-Stimulated Ca ²⁺ Flux .

(A) Wnt5a-mediated inhibition is PTX insensitive. At 24 h post-transfection, 293 cells were pretreated with vehicle or 100 ng/ml PTX for 24 h. Cells were then treated with indicated Wnt proteins concomitantly with vehicle or PTX for an additional 24 h, and luciferase assay was performed.

(B) PTX is active in 293 cells as PTX pretreatment of cells inhibits LPA-induced Erk1,2 activation. The 293 cells were pretreated with 100 ng/ml PTX for 24 h and then treated with 10 μM LPA or vehicle for 10 min. Western blot analysis was then performed on total cell lysates using anti–phospho-Erk1,2 antibody. Membrane was stripped and reprobed for total Erk1,2 as a loading control.

(C) Wnt5a protein does not directly stimulate intracellular Ca ²⁺ flux. The 293 and 293Fz4 cells were loaded with Fura-2-dextran and then monitored for changes in intracellular Ca ²⁺ concentration as determined by the change in 340/380 excitation wavelength ratio following Wnt5a (500 ng/ml) and subsequent ionomycin treatment. Data represent the average 340/380 excitation wavelength ratio of three or more independent cells within the same field ± SD. Wnt5a has little effect on the ratio over the 15-min time course, whereas ionomycin treatment rapidly and robustly induces Ca ²⁺ flux in these cells. Prolonged treatment with Wnt5a (up to 1 h) did not result in changes in intracellular Ca ²⁺ levels (unpublished data).