Fig. 5. Smad2-P–Smad4–LEF1 directly inhibits E-cadherin gene expression. (A) Reporter gene analysis of E-cadherin promoter activity (pGL3-E-cad-Lux) demonstrated decreased expression under the influence of TGF3. Treatment of cells with dominant-negative Smad4 or LEF1 (DN Smad4 or DN LEF1, respectively) prevented the repression of E-cadherin, but antisense -catenin/-catenin oligodeoxynucleotide (AS B-catenin ODN) did not (mean ± s.d.; n=3; ^*P<0.001 compared with Unfused; ^**P<0.05 compared with E-cad). (B) Site directed mutagenesis of the Smad4-binding (SBE) and LEF1-binding (E-pal and non E-pal) regions also prevented loss of E-cadherin promoter activity (mean ± s.d.; n=3; ^*P<0.001 compared with Unfused; ^**P<0.01 compared with E-cad). (C) Real-time quantitative PCR for E-cadherin gene expression relative to the unfused MEE negative control showed a steady decrease in fused MEE cells and fused cells treated with TGF3. DN-LEF1 and DN Smad4 prevented this suppression, whereas AS B-catenin ODN had no effect (mean ± s.d.; n=3; ^*P<0.001 compared with Unfused; ^**P<0.01 compared with + TGF3).