Figure 4

Effect of Slug knockdown on the expression of specific genes. MDA-MB-231 cells were transfected with si-Slug molecule or a non-relevant siRNA (si-Scr). (A) E-cadherin, ERα, TRPS1 expression was determined at mRNA level, and revealed by quantitative RT-PCR analysis. Data are represented as fold change respect to control sample (Ctr) for each gene analysed. Results represent means ± SEM of three independent experiments. p-values ≤ 0.05 were considered statistically significant. (B) ERα, p53, Vimentin, and E-cadherin expression was determined at protein level, and revealed by Western Blot. (C) Analysis of the 2 Kb in size promoter region of Slug, ERα, p53, Vimentin, E-cadherin and TRPS1 genes. Predicted E-boxes consensus-binding site are indicated with grey ovals. (D) Slug is recruited at TRPS1 promoter in vivo. The localization of predicted Slug consensus binding sites (5'-CAGGTG-3' or 5'-CACCTG-3') in the human TRPS1 promoter is reported. Protein-DNA complexes were in vivo formaldehyde-cross linked in MDA-MB-231 cells. Chromatin fragments were subjected to immunoprecipitation with antibody against endogenous Slug. A negative control using nonspecific normal rabbit antibody against Ig λ chain was also included. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the primers pairs spanning the reported regions of TRPS1 promoter (PCR amplicons are indicated by horizontal bars). Aliquots of chromatin taken before immunoprecipitation were used as Input positive controls whereas chromatin eluted from immunoprecipitation lacking antibody was used as no antibody control (No Ab). All experiments were repeated at least three times and representative images shown.