Volume 138, Issue 2, Pages 627-635 (February 2010) CDX Transcription Factors Positively Regulate Expression of Solute Carrier Family 5, Member 8 in the Colonic Epithelium  Fumihiko Kakizaki, Koji Aoki, Hiroyuki Miyoshi, Nancy Carrasco, Masahiro Aoki, Makoto M. Taketo  Gastroenterology  Volume 138, Issue 2, Pages 627-635 (February 2010) DOI: 10.1053/j.gastro.2009.10.047 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 ChIP-PCR analysis of Cdx1 and Cdx2 binding to the SLC5A8 promoter. (A) Anti-Flag ChIP using extracts of DLD-1 and HCT116 transfected with vector alone (Control), Flag-Cdx1, or Flag-Cdx2. The promoter regions of SLC5A8 and the known CDX2 target genes CDH17 and SI were PCR amplified. ACTB was used as a negative control. In lanes 7–9 (ChIP+FP), the anti-Flag antibodies were blocked by an excess amount of the Flag peptide. (B) ChIP against endogenous CDX1 and CDX2 was also performed using extracts of T84 cells. CDH17 was used as a positive control. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Transcriptional activation of SLC5A8 promoter by Cdx1 and Cdx2. (A) Schematic representation of the luciferase reporter gene constructs containing the SLC5A8 promoter fragments. “−1344/+53” Is the wild-type SLC5A8 promoter-reporter construct. “−853/+53” and “−262/+53” are deletion mutants that lack the ChIP fragment region. “−1344/+53 mut” contains point mutations that abolish CDX binding at the 4 CDX binding consensus sequences of “−1344/+53.” Arrowheads: positions of the CDX binding consensus sequences; Gray bar: the ChIP fragment region. (B) Luciferase reporter gene assays for Cdx-mediated activation of the SLC5A8 promoter. DLD-1 (left) or HCT116 (right) cells were transfected with a reporter construct and an expression vector for Flag-Cdx1 or Flag-Cdx2. Luciferase activities of the control vector-transfected cells were assigned the value of 1. *P < .01 and #P < .01 compared with “−1344/+53” values for Cdx1 and Cdx2, respectively. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Induction of the endogenous SLC5A8 by Cdx1 and Cdx2. (A and B) QRT-PCR analyses for induction of the endogenous SLC5A8 expression by overexpression of Flag-Cdx1 or Flag-Cdx2 in DLD-1 cells using Tet-Off system. *P < .01 compared with control. (C) QRT-PCR analyses for induction of the endogenous SLC5A8 expression in HCT116 cells transiently transfected with a Flag-Cdx1 or Flag-Cdx2 expression vector. *P < .01 compared with control. (D) Western blot analyses for induction of the SLC5A8 protein by Flag-Cdx1 or Flag-Cdx2 in HCT116 cells. GAPDH was used as a loading control. (E) Immunofluorescence analysis for expression of SLC5A8 in HCT116 cells overexpressing Cdx1 or Cdx2. Green: SLC5A8. Blue: DAPI stained nuclei. Scale bars, 20 μm. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Reduction of the endogenous SLC5A8 level by CDX1 and/or CDX2 knockdown. T84 cells were transfected with siRNA oligonucleotides against CDX1 and/or CDX2 as described in Supplementary Methods. (A) Western blot analysis for CDX1 and CDX2 levels in siRNA-treated T84 cells. GAPDH was used as a loading control. (B) QRT-PCR analysis of the SLC5A8 levels in siRNA-treated cells. The SLC5A8 level in control siRNA-treated cells was assigned the value of 1. *P < .05; **P < .01. (C) Western blot analysis for SLC5A8 expression in T84 cells treated with siRNAs against CDX1 and CDX2. (D) Immunofluorescence analysis for expression of SLC5A8 in CDX1/2 double-knockdown T84 cells. Green: SLC5A8. Blue: DAPI stained nuclei. Scale bars, 20 μm. Control, 30 nmol/L control siRNA; siCDX1, 20 nmol/L control siRNA plus 10 nmol/L CDX1 siRNA; siCDX2, 10 nmol/L control siRNA plus 20 nmol/L CDX2 siRNA; siCDX1+siCDX2, 10 nmol/L CDX1 siRNA plus 20 nmol/L CDX2 siRNA. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Effects of overexpression or knockdowns of CDX transcription factors on Na+-dependent uptake of nicotinate. Analysis of [14C]-nicotinate uptake in human colon cancer cells. (A) HCT116 cells were transiently transfected with either vector alone (Control), Flag-Cdx1, or Flag-Cdx2, and the Na+-dependent uptake of [14C]-nicotinate (5 μmol/L) at 37°C was measured at 2 minutes. (B) T84 cells were transfected with control siRNA (Control), siRNA against CDX1 and siRNA against CDX2 (siCDX1+siCDX2), and the Na+-dependent niotinate uptake was measured as in A. *P < .05 compared with control. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Expression of Cdx1, Cdx2, and Slc5a8 in mouse colon. (A) RT-PCR analysis for expression of Cdx1, Cdx2, and Slc5a8 in the epithelial and stromal compartments of the mouse colon. RNA was prepared from each layer collected by laser microdissection. Krt19 (encoding keratin 19), epithelial cell marker; Vim (encoding vimentin), stromal cell marker. (B) Immunofluorescence analysis for localization of Cdx1, Cdx2, and Slc5a8 in the mouse colonic tissue sections. H&E, hematoxilin and eosin staining. Slc5a8/Cdx2, double immunofluorescence staining of Slc5a8 (green) and Cdx2 (red). Cdx1/Cdx2, double immunofluorescence staining of Cdx1 (green) and Cdx2 (red). Scale bar, 20 μm. (C) Nuclear Cdx1 and Cdx2 expression in the proximal-distal axis of the mouse colon. (D) Expression of Slc5a8 mRNA in the proximal-distal axis of the mouse colon. cDNA from the kidney was used as a positive control. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Expression of Slc5a8 in the colon of Cdx1−/− and Cdx2+/− mice. (A) QRT-PCR for Slc5a8 expression in the colon of Cdx1+/+ and Cdx1−/− mice. Results are given as means ± SD (n = 5). *P < .01 compared with Cdx1+/+ mice. (B) Expression of Slc5a8, Cdx1, and Cdx2 in the colon of Cdx1+/+ and Cdx1−/− mice analyzed by Western blot. Actb was used as a loading control. (C) QRT-PCR analysis for expression of Slc5a8 mRNA in the colon of Cdx2+/− mice. Results are given as mean ± SD (n = 5). *P < .01 compared with Cdx2+/+ mice. (D) Western blot analysis for expression of Slc5a8, Cdx1, and Cdx2 in the colon of Cdx2+/+ and Cdx2+/− mice. (E) QRT-PCR for Slc5a8 expression in the hamartoma and adjacent normal tissues in the proximal colon of Cdx2+/− mice. N, normal tissue; T, tumor. Results are given as mean ± SD (n = 3). *P < .01 compared with normal proximal colon. (F) Western blot analyses for expression of nuclear Cdx1 and Cdx2 in the hamartomas and adjacent normal tissues in the colon of Cdx2+/− mice. N, normal tissue; T, tumor. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 Expression of Cdx1, Cdx2, and Slc5a8 in the colonic polyps of Apc+/Δ716 mice. (A) Western blot analysis for expression of nuclear Cdx1 and Cdx2 in the polyps and adjacent normal tissues of Apc+/Δ716 mice. Total histone H3 used as a loading control. (B) QRT-PCR analysis for Slc5a8 mRNA expression in the colonic polyps of the Apc+/Δ716 mouse. Results are given as mean ± SD (n = 3). *P < .01 compared with normal distal colon control. (C) Immunohistochemistry analysis displaying the loss of Slc5a8 protein in a colonic polyp of Apc+/Δ716 mice. Scale bar, 200 μm. Dashed line indicates the boundary between normal tissue and adenoma. N, normal tissue; Ad, adenomatous polyp. Gastroenterology 2010 138, 627-635DOI: (10.1053/j.gastro.2009.10.047) Copyright © 2010 AGA Institute Terms and Conditions