Volume 137, Issue 2, Pages 629-638 (August 2009) Reduced Level of Smoothened Suppresses Intestinal Tumorigenesis by Down- Regulation of Wnt Signaling  Sumimasa Arimura, Akihiro Matsunaga, Takanori Kitamura, Koji Aoki, Masahiro Aoki, Makoto M. Taketo  Gastroenterology  Volume 137, Issue 2, Pages 629-638 (August 2009) DOI: 10.1053/j.gastro.2009.04.059 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Expression of Smo in intestinal polyps of ApcΔ716 mice. (A) Expression of Smo and Hh target genes analyzed by quantitative RT-PCR in the small intestinal (left) and colonic (right) polyps of the ApcΔ716 mice. Error bars indicate SD (n = 4). *P < .05; **P < .01. (B) Expression of Smo and β-actin (control) analyzed by Western blotting in the intestinal polyps of the ApcΔ716 mice. (C) Expression of Smo analyzed by RT-PCR in cDNA prepared from laser-microdissected adenoma cells of the ApcΔ716 mouse intestinal polyps. Whole, whole adenoma tissues; Epithel, laser-microdissected adenoma epithelial cells. (D) Expression of Smo analyzed by in situ hybridization in the small intestinal (a, b, e, f) and colonic (c, d, g, h) polyps of the ApcΔ716 mice. Scale bars, a and e: 50 μm; c and g: 30 μm; b, d, f, h: 5 μm. Letters A, adenoma; E, normal epithelium; S, stroma. Gastroenterology 2009 137, 629-638DOI: (10.1053/j.gastro.2009.04.059) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Effects of SMO down-regulation by siRNAs on cell proliferation in human colon cancer cell lines. (A) Expression of SMO and GAPDH (control) analyzed by RT-PCR and Western blotting in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours. (B) Growth curves of the SW480 and HCT116 cells treated with SMO siRNAs for 96 hours, determined in a Coulter counter. (C and D) Cell cycle distribution (C) and percentages of the sub-G1 populations (D) in flow-cytometric analyses of the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours. (E) Expression of cell cycle regulators involved in the G1/S progression analyzed by Western blotting in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours. Error bars indicate SD. *P < .05; **P < .01. N.T., no treatment with siRNA; siN.S., 40 nmol/L nonsilencing siRNA; siSMO, 40 nmol/L siRNAs directed against SMO gene. Gastroenterology 2009 137, 629-638DOI: (10.1053/j.gastro.2009.04.059) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Effects of Smo heterozygosity on intestinal tumor growth in Apc mutants. (A) Expression of Smo and β-actin analyzed by Western blotting in the small intestinal polyps of the ApcΔ716 and ApcSmo mice. (B and C) Total polyp number per mouse (B) and size distribution of the intestinal polyps (C) in the small intestine of the ApcSmo and littermate ApcΔ716 mice. (D) Dissection micrographs of the intestines from the ApcΔ716 and ApcSmo mice. Polyps visible here are in the large size class (Φ ≥ 1–2 mm). Duo, duodenum; Jej, jejunum; Ile, ileum. Scale bars, 1 cm. (E) Dissection micrographs and H&E-stained sections of the intestinal mucosa containing well-developed ileal polyps in the ApcΔ716 and ApcSmo mice. Scale bars, 200 μm (red) and 100 μm (black). Arrowheads indicate polyps. (F) Staining of the adenoma cells with BrdU in the small intestinal polyps of ApcΔ716 and ApcSmo mice (top). Quantified data for BrdU-positive adenoma cells (bottom). Scale bars, 50 μm. Error bars indicate SDs (n = 9 for B and C, and 6 for F). *P < .05; **P < .01. Gastroenterology 2009 137, 629-638DOI: (10.1053/j.gastro.2009.04.059) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Effects of SMO down-regulation on GLI-dependent transcription in SMO-high human colon cancer cell lines and in the polyps of ApcΔ716 mice. (A) Luciferase reporter assays using 8×3′Gli-BS-Luc and 8×mut3′Gli-BS-Luc reporter plasmid in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours. (B and C) Expression of SMO and the Hh target genes analyzed by quantitative RT-PCR in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours (B) and in the small intestinal and colonic polyps of ApcΔ716 and ApcSmo mice (C). Error bars indicate SD. **P < .01. N.T., no treatment with siRNA; siN.S., 40 nmol/L nonsilencing siRNA; siSMO, 40 nmol/L siRNAs directed against SMO gene; ND, not detectable. Gastroenterology 2009 137, 629-638DOI: (10.1053/j.gastro.2009.04.059) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effects of SMO down-regulation on β-catenin-dependent transcription in SMO-high human colon cancer cell lines and in the polyps of ApcΔ716 mice. (A) Luciferase reporter assays using TOPFLASH and FOPFLASH reporter plasmids in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours. (B and C) Expression of SMO and the Wnt target genes analyzed by quantitative RT-PCR in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours (B) and in the small intestinal and colonic polyps of ApcΔ716 and ApcSmo mice (C). Error bars indicate SD. *P < .05; **P < .01. N.T., no treatment with siRNA; siN.S., 40 nmol/L nonsilencing siRNA; siSMO, 40 nmol/L siRNAs directed against SMO gene; ND, not detectable. Gastroenterology 2009 137, 629-638DOI: (10.1053/j.gastro.2009.04.059) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effects of SMO knockdown on active β-catenin in SMO-high human colon cancer cell lines and in the polyps of ApcΔ716 mice. (A and B) Expression of total and active β-catenin analyzed by Western blotting in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours (A) and in the intestinal polyps of ApcΔ716 and ApcSmo mice (B). (C) Immunofluorescence analysis for subcellular localization of active β-catenin in the SW480 and HCT116 cells treated with SMO siRNAs for 72 hours. N.T., no treatment with siRNA; siN.S., 40 nmol/L nonsilencing siRNA; siSMO, 40 nmol/L siRNAs directed against SMO gene. Gastroenterology 2009 137, 629-638DOI: (10.1053/j.gastro.2009.04.059) Copyright © 2009 AGA Institute Terms and Conditions