SRF directly induced Snail expression and transcriptional activation in HPMCs.

<p>(a) Identification of SRE1 and SRE2 in the SNAIL promoter. Chromatin immunoprecipitation was used to examine SRF binding to the SNAIL promoter in HG-induced HPMCs and the control. Reaction controls included immunoprecipitations performed using a nonspecific IgG monoclonal antibody; PCR was performed using whole cell genomic DNA (Input). A representative example of three independent experiments is shown. (b) EMSA was used to analyze the SRF-binding site induced by HG. (b1) Samples 1 to 7 were induced by HG for 0 h, 5 h, 18 h, 72 h, and 96 h and compared with a positive control and negative control. SRE was bound after inducing for 72 h. (b2) Analysis of the SRF-binding site in the SNAIL promoter by EMSA. Biotin-labeled SRE1 and SRE2 oligonucleotides were used as a probe. Of the two examined putative SRE-binding sites within the SNAIL gene promoter, only SRE2 displayed specific SRF binding in HPMCs induced by HG. Samples 1 to 7 were 72 h+SRE, 72h+SRE + cold-SRE, 72h+SRE + cold SER Mut, 72h+SRE1, 72 h+SRE1+ cold-SRE1, 72h+SRE1+ cold-SRE1 Mut, 72h+SER2, 72h+SER2 +cold-SRE2, and 72h+SER2 +cold-SRE2 Mut. Cold probe, unlabeled SRE; SRE mut, mutated SRE. (c) For the competition assays with SRE1 and SRE2, 200-fold molar excess of the Snail plasmid and mutant SRE1, deleted SRE1, mutant SRE2, deleted SRE2 and mutant SRE1 and SRE2 were used in HPMCs (c1) and HG-induced HPMCs (c2). All results shown are representative of at least three independent experiments. * P<0.05 vs. Snail (SRE1+SRE2) plasmid; ** P<0.05 vs. control plasmid.