(A) Representative photomicrographs of IHC staining for SOX9 in normal human colon tissue showing nuclear SOX9 expression in crypt base cells. The image is shown with low magnification for the entire colon crypts; boxed areas for lumen and crypt base regions are also shown as high magnification (middle and bottom panels). (B–F) Representative photomicrographs of IHC staining for SOX9 in CRCs in a human tissue microarray, with different IHC scores of (B) 0, (C) +/–, (D) 1+, (E) 2+, and (F) 3+. The images with low magnification (left) and their corresponding boxed areas with high magnification (right) are displayed for each CRC. Scale bars: 100 μM (low magnification), 20 μM (high magnification). (G) Immunoblot analysis for SOX9 in human colon cancer cell lines, with β-actin as a loading and transfer control. (H) Diagram of CpG islands in the SOX9 promoter and first exon, and locations of primers for bisulfite sequencing (F1/R1, F2/R2, F3/R3). Lollipop diagrams of bisulfite sequencing of SOX9 at the indicated regions (relative to the +1 transcription start site) in HT29 and RKO cells are shown with black lollipops for methylated CpGs and white for unmethylated. At least 5 clones were sequenced for each primer pair, and methylation of an individual CpG dinucleotide was confirmed in at least 2 clones. (I) RKO cells were treated with vehicle (0) or treated with 1 μM or 5 μM 5-Aza-2′-deoxycytidine (5-AzaD) for 3 days to induce DNA demethylation. During the third day of treatment with vehicle or 5-AzaD, cells were further incubated with vehicle or 0.5 μM trichostatin A (TSA) for 24 hours. SOX9 expression in the cells was assessed by immunoblot, with β-actin as a loading and transfer control.