Figure 1.

Snail and Slug promote β-catenin–LEF-1 activity. (A) Immunocytochemistry demonstrating translocation of β-catenin to the nuclei of DLD1 cells transfected with either Snail or Slug expression plasmids, as well as increased expression and nuclear localization of LEF-1. Bar, 10 μm. (B) Immunoblotting confirming increased expression of Snail, Slug, and LEF-1 in response to the Snail and Slug constructs. (C and D) pTOPFLASH-Lux reporter assay assessing increased LEF-1 transcriptional activity upon cotransfection with Snail or Slug expression plasmids. Addition of DN Smad4 and DN LEF-1 adenoviral constructs significantly inhibited luciferase activity. Data represent mean±SD; n=3; *p<0.01 compared with control; **p<0.05 compared with Snail or Slug. (E) Real-time quantitative PCR analysis showing reduction of E-cadherin expression in cells exposed to Snail or Slug plasmids. These inhibitions were mostly prevented by DN LEF-1. (F) Biochemical confirmation of EMT by immunoblotting, showing decreased E-cadherin expression, as well as increased vimentin and fibronectin expression in the presence of the Snail or Slug constructs. Addition of a DN LEF-1 plasmid prevented these changes. (G) Phase contrast imaging showing the EMT phenotype in Snail- or Slug-treated cells, which is prevented by DN LEF-1. (H) Cell invasion/migration assays observing increased levels in cells transfected with Snail or Slug plasmids. Addition of DN LEF-1 prevented these increases. Data represent mean±SD; n=3; *p<0.0001 compared with control.