Figure 2

N-cadherin functions in Wnt-induced β-catenin signaling in a cell-autonomous manner. (A) Separate populations of 293 T cells were transfected with Wnt3a expression and pSuper8TOPFlash reporter constructs and plated either on the opposite of a polycarbonate transwell filter (“membrane bound”) or on the bottom of the transwell assay (“separated”). In a TOPFlash reporter assay, Wnt3a-expressing signaler cells induced β-catenin signaling in reporter cells adherent on the opposite face of the transwell membrane, but not in reporter cells plated separately at the bottom of the chamber. P = 0.004 by repeated measures analysis of variance (ANOVA), ***P < 0.001, Neuman Keuls post-hoc test, n = 3. (B) Reduction of N-cadherin by shRNA (Ncad-shRNA) or by overexpression of C-terminal truncated N-cadherin (NcadΔC) in Wnt-responsive cell results in reduced Wnt-activated β-catenin transcriptional activation. Wnt3a transfected signaler cells were co-cultured with pTOPflash-transfected reporter cells co-transfected with either shRNA to N-cadherin (P = 0.0012 by repeated measures ANOVA, **P < 0.01, *P < 0.05 by Neuman Keuls post-hoc test; n = 4) or NcadΔC (P = 0.0006 by repeated measures ANOVA, ***P < 0.001, **P < 0.01 by Neuman Keuls post-hoc test; n = 3), and luciferase activity was measured 24 hours after co-culture. (C) Inhibition of N-cadherin in the Wnt-producing signaling cell does not affect Wnt-mediated β-catenin signaling. Wnt3a transfected signaler cells were co-transfected with either Ncad-shRNA (P = 0.0024 by repeated measures ANOVA, **P < 0.01 by Neuman Keuls post hoc test; n = 4) or NcadΔC (P = 0.0002 by repeated measures ANOVA, ***P < 0.001 by Neuman Keuls post-hoc test; n = 3), co-cultured with pTOPflash-transfected reporter cells, and luciferase activity was measured 24 hours after co-culture.