Modulation of Wnt signaling by soluble Fz8-CRD-Fc. (A) Wnt reporter assay showing the influence of purified mFz8-CRD-Fc on Wnt signaling in a dose-dependent manner. (B) Immunoblot depicting the influence of recombinant Fz8-CRD-Fc on the concentration of soluble Wnt3a protein in the cell supernatant. The non-binding Wnt3a mutant W333A was used as a control. For all conditions, cells were seeded at equal cell density. (C) Double luciferase chamber assay using CMV-β-galactosidase and Super TOPFlash reporter in HEK293 cells. The Fz8-CRD-Fc protein was applied at increasing concentrations to the Wnt-producing cells. Luciferase activity was monitored in Wnt3a responder cells. Inset shows the set-up of the chamber assay. Experiments were carried out in triplicate. Error bars depict standard deviation. Statistical significance in relative luciferase activity levels compared to the wild-type Wnt3a levels (A, C) as indicated: *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. as not significant according to Student’s t-test. CRD, cysteine-rich domain; WB, Western blot.