Figure 2.
SNAIL1 is directly involved in the regulation of E-cadherin expression and nuclear translocation of β-catenin in DNMT1ΔE3−6 cells. (A) Immunolocalization analyses showing re-expression of E-cadherin and relocalization of β-catenin from the cell nucleus to newly formed cell–cell contacts after siRNA-mediated depletion of SNAIL1 in DNMT1ΔE3−6 cells. Stable transfections of shRNA-EGFP or Mock vectors were performed as controls. At least three stable clones or transient transfections were analyzed. Bar: 10 μm. (B) siRNA-mediated depletion of SNAIL1 in cells expressing DNMT1ΔE3−6 by stable transfection of the corresponding shRNAs. Protein expression by immunoblot indicates that E-cadherin is re-expressed after depletion of SNAIL1. Similarly, levels of a metabolically stabilized form of β-catenin, dephosphorylated on Ser37 or Thr41 (β-catenin-ABC), are strongly reduced after SNAIL depletion in DNMT1ΔE3−6 cells. No significant changes of DNMT1 protein expression levels were observed. Three different siRNA-transfected clones are represented. β-tubulin expression was used as loading control. (C) Normalized Luciferase/Renilla activities in HCT116 DNMT1ΔE3−6 control and siSNAIL1 cells, after transient cotransfection of reporter vectors containing multimerized promoter sequences recognized by β-catenin-Lef/Tcf complexes (upper) or cyclin D1 promoter (lower). Analyses were performed in triplicate and the mean ± SD are shown. **P < 0.001.