1. Volume 143, Issue 4, Pages 1037-1047 (October 2012)
β-Catenin Regulates Deiodinase Levels and Thyroid Hormone Signaling in Colon Cancer Cells 
Monica Dentice, Cristina Luongo, Raffaele Ambrosio, Annarita Sibilio, Antonella Casillo, Antonino Iaccarino, Giancarlo Troncone, Gianfranco Fenzi, P. Reed Larsen, Domenico Salvatore 
Gastroenterology 
Volume 143, Issue 4, Pages (October 2012)
DOI: /j.gastro
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2. Figure 1
Wnt/β-catenin pathway regulates type 2 and 3 deiodinase expression. (A) CaCo-2 cells were plated at 60% confluence (day 0) and harvested at the indicated days for reverse-transcription polymerase chain reaction (RT-PCR) analysis of D3 messenger RNA (mRNA) expression as indicated. Cyclophillin A was used as internal control. Western blot analysis of lysates from CaCo-2 cells spontaneously differentiated as indicated in A. Actin antibody was used as loading control. (B) RT-PCR and Northern blot analysis of D2 expression from the same samples as in A. (C and D) HCT-116 cells were transfected with a dominant negative TCF-4 (ΔN-TCF-4) or a control plasmid (CTR) and D3 and D2 mRNA measured at the indicated time. Values are mean ± standard error of mean of at least 3 independent experiments. (E) CaCo-2 cells were transfected at the indicated days with TRE3TK-Luc construct or TOP-Flash plasmid together with CMV-Renilla as internal control. The results are shown as means ± standard deviation of the LUC/Renilla ratios from at least 3 separate experiments, performed in duplicate. (F) Immunohistochemical analysis of D3 expression in mouse small intestine. Arrows indicate localization of D3 in intestinal crypts. Competition with a specific D3 peptide is also shown.*P < .05, **P < .01, ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
DIO3 promoter is a direct target of β-catenin/TCF-4 complex and physically associates with β-catenin. (A) Functional analyses of Dio3 promoter were carried out in HEK-293 cells. Dio3 promoter constructs were cotransfected into HEK-293 with constitutively active β-catenin (top) or TCF-4 (bottom) plasmids and empty vector. TOP-Flash and FOP-Flash constructs were used as positive and negative controls, respectively. (B, top) Electrophoretic mobility shift assay (EMSA) showing the ability of D3-C to form protein-DNA complexes with nuclear extracts from β-catenin-transfected cells. The specificity of the complex was verified by competition analysis with different competitors, ie, cMyc binding site (Myc) or 1 unrelated sequence (Unrel) (B, bottom). Alignment of the Dio3 promoter region containing the “D3-C” site from different mammalian species. The oligonucleotide used for EMSA, spanning the D3-C site (in gray) is underlined. (C) Chromatin immunoprecipitation was carried out to evaluate the in vivo binding of β-catenin to the DIO3 promoter. The amount of precipitated chromatin was calculated relative to the total input chromatin and expressed as percentage of the total.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

4. Figure 3
T3 treatment affects CaCo-2 cell proliferation and differentiation. (A) Growth curve of CaCo-2 cells cultured in 10% fetal bovine serum medium supplemented with vehicle or 30 nmol/L T3. (B) CaCo-2 cells treated with vehicle or 30 nmol/L T3 were incubated for 1 hour with [methyl-3H] thymidine. [3H] Thymidine incorporation in untreated cells was arbitrarily set as 100%. Values are mean ± standard error of mean of at least 3 independent experiments, *P < .05. (C) Proliferating CaCo-2 cells were treated with 30 nmol/L T3 for 48 hours, and total lysates were analyzed by Western blot for cyclin D1 expression. (D) Western blot analysis of proliferating (3 days) and differentiated (14 days) CaCo-2 cells treated with T3 or vehicle or transiently transfected with a dominant negative TRβ-1 plasmid (TRdn) or empty vector (CTR) as indicated. (E) Proliferating CaCo-2 cells were treated with vehicle or T3, and messenger RNA was analyzed for indicated genes by reverse-transcription polymerase chain reaction.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

5. Figure 4
Effects of T3 treatment on β-catenin subcellular localization, DNA-binding and transcriptional activity. (A) Subcellular localization of β-catenin was evaluated in CaCo-2 cells cultured in the presence or absence of T3 by subcellular fractionation as indicated. (B) The β-catenin/DNA binding capacity was evaluated by electrophoretic mobility shift assay (EMSA) using a radiolabeled c-Myc oligonucleotide and nuclear extracts from CaCo-2 cells treated with vehicle or T3 as indicated. (C) Proliferating CaCo-2 cells were transiently cotransfected with TOP-Flash promoter and treated with vehicle (CTR) or 30 nmol/L T3. The results are shown as means ± standard deviation of the LUC/Renilla ratios from at least 3 separate experiments, performed in duplicate. (D) Schematic diagram illustrating the proposed nuclear β-catenin-T3 homeostatic interactions and the role of D2 and D3 in CaCo-2 cells.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
D3-depletion reduces CaCo-2 cell proliferation and enhances differentiation. (A) CaCo-2 cells in the proliferating (day 3), intermediate (day 10), or differentiating phase (16 days) were transfected with TRE3TK-Luc promoter and treated with 5 mmol/L iopanoic acid (IOP) or vehicle (CTR) for 12 hours. The results are shown as means ± standard deviation of the LUC/Renilla ratios. **P < .01 compared with vehicle treated cells; n = 4. (B) Growth curve was performed in CaCo-2 cells cultured in 10% fetal bovine serum medium supplemented with vehicle or iopanoic acid. (C and D) CaCo-2 cells were transiently transfected with a D3 specific RNAi pool (iD3) or control scrambled oligonucleotides (iCTR) and E-cadherin and cyclin D1 expression levels measured by reverse-transcription polymerase chain reaction and Western blot analysis. Differentiated CaCo-2 cells (DIFF) were used as references for E-cadherin and cyclin D1 expression levels. (E and F) SW480 miRC− and miRD3 cells (4*106 cells/100 μL) were injected subcutaneously into the right and left flank of the nude mice. Tumor size was measured as described under Materials and Methods section. Each point represents the mean ± se of four mice. **P < .01; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
D3 protein expression is up-regulated in human colon cancer. (A and B) D3 expression was evaluated by immunohistochemistry (IHC) analysis in human normal colon (n = 41), colon adenomas (n = 13), and carcinomas (n = 51) tissues. (A) Statistical significance of the IHC analysis. The medium score as well as the overall range measured is indicated. (B) Representative D3 immunostaining from the indicated human samples.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

8. Figure 7
D3 is highly expressed in the early stages of human colon tumorigenesis. (A) D3 messenger RNA (mRNA) expression levels were evaluated in 48 complementary DNAs from colorectal cancer and matched normal mucosa. Actin mRNA levels were measured as an internal control. Results are shown as cancer sample/normal counterpart D3 expression. (B) Statistical analysis of D3-positive human colon tumors of different grading by immunohistochemical analysis was carried out using the Kruskal–Wallis test. (C) Representative images of D3 expression by immunohistochemical analysis of human colon cancer samples corresponding to different tumor grading at the indicated magnifications.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions