Volume 138, Issue 5, Pages 1863-1874.e1 (May 2010) Cooperation Between the Thyroid Hormone Receptor TRα1 and the WNT Pathway in the Induction of Intestinal Tumorigenesis  Elsa Kress, Seham Skah, Maria Sirakov, Julien Nadjar, Nicolas Gadot, Jean–Yves Scoazec, Jacques Samarut, Michelina Plateroti  Gastroenterology  Volume 138, Issue 5, Pages 1863-1874.e1 (May 2010) DOI: 10.1053/j.gastro.2010.01.041 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Generation of vil-TRα1 animals. (A) TRα1 protein immunolabeling in paraffin sections from wild-type (WT) or vil-TRα1 mice. Images show merged TRα1 immunolabeling (red) and nuclear staining (blue) in small intestine and colon. (B) Morphological analysis of small intestine and colon from WT and vil-TRα1 mice. c, crypts; v, villi; sm, smooth muscle; se, surface epithelium. (C) Ki-67 immunolabeling of proliferating cells in intestinal sections from WT or vil-TRα1 mice. Images show merged Ki-67 immunolabeling (red) and nuclear staining (blue) in small intestine and colon. (D) Quantification of the proliferating cells along the proximo-distal intestinal axis. The Ki-67–positive cells have been counted under a Zeiss Axioplan microscope on well-oriented sections from 4 different animals per genotype. Thirty crypts per condition have been evaluated under the microscope. Numeric values illustrate mean ± standard deviation. Dotted bars in (A) and (C) define the limit between the crypts and the villi. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 The vil-TRα1 mice display a high level of β-catenin and WNT activation. (A) β-catenin immunolabeling in intestinal paraffin sections from WT or vil-TRα1 mice. Images show merged β-catenin immunolabeling (red) and nuclear staining (blue) in small intestine and colon. Bars = 15 μm; enlargements = 5 μm. (B) Western blot analysis (50 μg protein/lane) confirming the increased levels of total and activated β-catenin in vil-TRα1 mice compared with WT. The activation of WNT is confirmed by increased amounts of c-Myc. The image is representative of 3 independent experiments. Actin has been used as loading control. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Analysis of vil-TRα1/Apc mice. (A) H&E-stained sections of small intestine and colon from 3-month-old Apc and vil-TRα1/Apc. (B) Analysis of cell proliferation by Ki-67 immunolabeling in small intestine and colon of 3-month-old Apc and vil-TRα1/Apc mice. Images show merged Ki-67 immunolabeling (red) and nuclear staining (blue). (C) H&E-stained sections of small intestine and colon from 6-month-old Apc and vil-TRα1/Apc as indicated. (D) Analysis of cell proliferation by Ki-67 immunolabeling in small intestine and colon of 6-month-old Apc and vil-TRα1/Apc mice. Images show merged Ki-67 immunolabeling (red) and nuclear staining (blue). (E) Presence of micrometastasis in the liver of vil-TRα1/Apc mice. Images show merged villin (red) and nuclear staining (blue). We analyzed 3 animals per genotype and the micrometastasis were present in the liver of all the double transgenic animals. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Activation of β-catenin in the intestine of vil-TRα1/Apc. (A–C) β-catenin immunolabeling in paraffin sections from Apc or vil-TRα1/Apc mice in normal mucosa (A), in tumors from 3-month-old vil-TRα1/Apc mice (B) or 6-months-old (C) animals of indicated genotype. Images show merged β-catenin immunolabeling (red) and nuclear staining (blue) in small intestine and colon as indicated. Bars = 30 μm; enlargements = 7 μm. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Western blot analysis of total and activated β-catenin. (A, B) The study was performed in the intestine (50 μg protein/lane) of 3- (A) and 6-month (B) animals. The picture is representative of 3 independent experiments. Actin has been used as loading control. N, normal mucosa; A, adenoma; AC, adenocarcinoma. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Analysis of WNT target genes in vil-TRα1/Apc mice. (A) Real-time quantitative polymerase chain reaction analysis of Ccnd1 and c-Myc in the intestine of 3- and 6-month-old mice. Values represent fold change ± standard deviation, after normalization to wild-type (WT). A 2-tailed Student t test was used for statistical analysis. *P < .05 and **P < .001, in comparison with WT. $P < .05 and $$P < .001, in comparison with healthy tissue of the same genotype; n = 4. (B) Western blot analysis (50 μg protein/lane) of cyclin D1 and c-Myc expression in the intestine of 3- and 6-month animals. The picture is representative of 3 independent experiments. Actin has been used as loading control. N, normal mucosa; A, adenoma; AC, adenocarcinoma. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Analysis of c-Fos in vil-TRα1/Apc mice. (A) Real-time quantitative polymerase chain reaction analysis of c-Fos in the intestine of 3- and 6-month-old mice. Values represent fold change ± standard deviation after normalization to wild-type (WT). A 2-tailed Student t test was used for statistical analysis. *P < .05 and **P < .001, in comparison with WT. $P < .05 and $$P < .001, in comparison with the normal mucosa of the same genotype; n = 4. (B) c-Fos immunolabeling in paraffin sections from Apc or vil-TRα1/Apc mice. Images show merged c-Fos immunolabeling (red) and nuclear staining (blue) in healthy small intestine, healthy colon, lesions from small intestine, or lesions from colon as labeled. c, crypt; v, villus; sm, smooth muscle; se, surface epithelium; lp, lamina propria. Bars = 15 μm. (C) Western blot analysis (50 μg protein/lane) confirming the differential expression of c-Fos in 3- and 6-month-old animals of different genotypes. The image is representative of 3 independent experiments. Actin has been used as loading control. N, normal mucosa; A, adenoma; AC, adenocarcinoma. Gastroenterology 2010 138, 1863-1874.e1DOI: (10.1053/j.gastro.2010.01.041) Copyright © 2010 AGA Institute Terms and Conditions