FIGURE 6.

Dapper3 inhibits Wnt signaling. A, shown is immunofluorescence staining of E-cadherin, α-SMA, and fibronectin in renal tubular cells. Primary renal tubular epithelial cells from Dapper3 wild-type and KO mice were treated with 40 ng/ml Wnt3a for 72 h and stained for E-cadherin (red), α-SMA (green), and fibronectin (green). Nuclei were counter-stained with DAPI (blue). Scale bars, 50 μm. B and C, Dapper3 inhibits Wnt3a (B)- or Dvl2 (C)-activated expression of TopFlash-luciferase reporter in a dose-dependent manner. HEK293T cells were co-transfected with reporter plasmid (0.1 μg), and the constructs encoding Wnt3a (0.1 μg) or Dvl2 (0.1 μg) with or without Dapper3 (50–200 ng). At 36 h post-transfection, the cells were harvested for luciferase assay. Experiments were repeated in triplicate. D, Dapper3 interacts with Dvl2. HEK293T cells were transfected with HA-Dapper3 and FLAG-Dvl2 as indicated. At 36 h post-transfection, the cells were harvested for anti-HA immunoprecipitation (IP) and anti-FLAG immunoblotting (IB, upper panel, arrow). Protein expression was confirmed by immunoblotting with the total cell lysates (middle and lower panels). The asterisk indicates IgG heavy chain. E, subcellular co-localization of exogenous Dapper3 and Dvl2 is shown. HeLa cells were transfected with FLAG-Dvl2 together with Myc-Dapper3. Subcellular localization of Dvl2 (red) or Myc-Dapper3 (green) was detected by indirect anti-FLAG or anti-Myc immunofluorescence. Their co-localization was shown in the merged images (yellow), and the nuclei were counter-stained with DAPI (blue). Scale bars, 10 μm. F, Dapper3 could effectively reduce the protein level of Dvl2. HEK293T cells were co-transfected with FLAG-Dvl2 (0.2 μg) and gradient Myc-Dapper3 (0.3–1.0 μg) along with GFP (0.2 μg) as a control. At 36 h post-transfection, the cells were harvested for immunoblotting.