Figure 3

In vivo recruitment of Slug protein at the miR-221-222 locus. (A) The localization of predicted Slug consensus binding sites (5'-CAGGTG-3' or 5'-CACCTG-3') in the human miR-222/221 locus region is indicated with grey ovals. Protein-DNA complexes were in vivo formaldehyde-cross linked in MDA-MB-231. Chromatin fragments were subjected to immunoprecipitation with antibodies against endogenous Slug and Acetyl Histone H3 (Ac-H3). 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 miR-221 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. MDA-MB-231 cells were transfected with 30 nM si-Slug molecule or a non-relevant siRNA (si-Scr). Pri-miR-221 (B) and miR-222 (C) expression levels were determined at RNA level after 72 h of treatment, and revealed by quantitative RT-PCR analysis. RT-PCR results were calculated using the ΔΔCt method and data are presented as fold change respect to untreated cells (Ctr). Results represent means ± SEM of three independent experiments. p-values ≤ 0.05 were considered statistically significant.