Figure 3.

Merlin inhibits Wnt/β-catenin signaling upstream of β-catenin. (a) Ectopic expression of Merlin is associated with reduced Wnt3a-mediated reporter activity. pSuperTop and pRL-TK reporter plasmids with Flag-Merlin as indicated were co-transfected into HEK293T cells. Cells were harvested 24 h after transfection, and luciferase activity was measured by the dual luciferase assay system. Wnt3a-CM was added to cells about 20 h before harvesting cells. In all subsequent reporter assays, the same reporter plasmids and assay system were used. Data represent average values from one representative experiment performed in triplicate. Error bars indicate standard deviations of triplicate. (b) Unphospho- or phosphor-mimetic forms of Merlin showed stronger or weaker inhibitory activity, respectively, than wild-type Merlin as determined by VSVG-LRP6ΔN-induced luciferase activity (left panel). Ectopic expression of Flag-Merlin was examined by western blotting (right panel). (c) Mutant form of Merlin (Merlin-K79E in NF2 patients) did not inhibit VSVG-LRP6ΔN-induced luciferase activity (left panel). Ectopic expression of Flag-Merlin was examined by western blotting (right panel). (d and e) Merlin inhibited Wnt3a-CM or Dvl but not β-catenin or the stabilized form of β-catenin as determined by reporter activity. Relative luciferase activities were measured in cells transfected with the indicated plasmids. Cells were treated with Wnt3a-CM for 20 h. Knockdown of Merlin was examined by western blotting (right panel). (f) Knockdown of Merlin enhanced expression of Wnt target genes. siGFP or siMerlin-transfected HEK293T cells were treated with L-CM or Wnt3a-CM overnight. Real-time PCR was performed to measure expression of Wnt target genes (Lef1 and c-Jun) and Merlin