Figure 5

Oct4/Nanog-mediated Stat3 activation regulates snail expression in 97 L-ON cells. (A) Association of Nanog with p-Stat3 in the nucleus of 97 L cell line. Equal amounts of protein were immunoprecipitated (IP) with an anti-Nanog monoclonal antibody or anti-p-Stat3-Y705 antibody and were immunoblotted to detect Nanog or p-Stat3 (Y-705). Normal mouse IgG was used as a control antibody. (B) Oct4/Nanog-induced Snail expression was significantly inhibited by a specific Stat3 inhibitor S3I-201 (10 μM for 48 h). (C) Knockdown of Stat3 reversed Oct-4/Nanog-induced overexpression of Snail in 97 L-ON cells. (D) Schematic representation of structure of Snail promoter reign. Grey rectangle represented putative Klf4 binding sites predicted using MatInspector. Black arrows (thin) depicted the location of the forward and reverse primers used for PCR amplification from immunoprecipitated DNA fragments. (E) Chromatin immunoprecipitation (ChIP) analysis of p-Stat3 enrichment in Snail promoter. A p-Stat3 antibody or IgG serum was conducted to immunoprecipitate DNA-protein complexes from 97 L-ON cells with p-Stat3 overexpression. IgG was used for a negative control. Binding of p-Stat3-containing transcription complex on the Snail promoter was enriched in 97 L-ON cells, compared with 97 L-Ctrol cell.