Fig. 5

TWIST1 regulates SOX2, CD44, GDF3, NANOG, MEIS1, and SALL4 gene expression through binding to their promoter region. Schematic representation of (A) SOX2, (B) CD44, (C) GDF3, (D) NANOG, (E) MEIS1, and (F) SALL4 promoter sequences indicating putative TWIST1-binding sites within a 2 kb region upstream of the TSS (+ 1). Arrows denote the canonical E-box consensus elements (CANNTG). The horizontal bars indicate the amplified promoter regions by PCR. G ChIP assays identified binding of the TWIST1 protein to the SOX2, CD44, GDF3, NANOG, MEIS1, and SALL4 proximal promoter regions. ChIP was performed in KYSE-30 cells expressing GFP + TWIST1, and was analyzed by PCR using promoter-specific primers. GAPDH was used as an internal reference gene for normalization. Anti-histone H3 antibody (ab1791, Abcam) was used as a positive control in ChIP. ChIP and PCR negative control samples consisted of no-antibody and no-chromatin, respectively. Genomic DNA from KYSE-30 cells expressing GFP + TWIST1 was used as a positive control for the input DNA. Gels were cropped for clarity. For the original gel images, see Fig. S2. H Band intensities of SOX2, CD44, GDF3, NANOG, MEIS1, and SALL4 PCR products for primer sets 1 and 2 were quantified using the Image J software, and the values were normalized to their corresponding control inputs, and the average relative band intensities are shown as bar graph. The results of triplicate experiments are shown as the mean ± S.D. (* p < 0.01, Student’s t-test). IP, immunoprecipitation; P1, Primer set 1; P2, Primer set 2