Gilles C., Polette M., Mestdagt M., Nawrocki-Raby B., Ruggeri P., Birembaut P. and Foidart J.M.
Notes: The authors explored the role of beta-catenin and T-cell factor 4 to transactivate vimentin, a protein involved in gain of mesenchymal characteristics and loss of epithelial characteristics as a precursor to metastasis. The vimentin promoter was cloned into the pGL3-Basic Vector. The beta-catenin/TCF binding sites upstream of a minimal c-fos promoter drove firefly luciferase expression in plasmids called TOP-FLASH (wild-type binding sites) and FOP-FLASH (mutant binding sites). To examine beta-catenin/TCF-4 induction, several human mammary epithelial cell lines were transfected with a mixture of 0.15µg of various firefly reporter constructs (wild type vimentin promoter, mutant vimentin promoter, TOP-FLASH,or FOP-FLASH), 0.15 µg of the beta-catenin expression vector (or the corresponding empty vector), 0.15 µg of the TCF-4 expression vector and 0.8 ng of the Renilla luciferase vector, phRG-TK. Twenty-four hours after transfection, the cells were lysed and assayed using the Dual-Luciferase® Reporter Assay System. (3087)