1. Volume 132, Issue 7, Pages 2412-2421 (June 2007)
Bone Morphogenetic Protein 4 Expressed in Esophagitis Induces a Columnar Phenotype in Esophageal Squamous Cells 
Francesca Milano, Jantine W.P.M. van Baal, Navtej S. Buttar, Agnieszka M. Rygiel, Floor de Kort, Cathrine J. DeMars, Wilda D. Rosmolen, Jacques J.G.H.M. Bergman, Jan van Marle, Kenneth K. Wang, Maikel P. Peppelenbosch, Kausilia K. Krishnadath 
Gastroenterology 
Volume 132, Issue 7, Pages (June 2007)
DOI: /j.gastro
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2. Figure 1
Expression of proteins of the BMP pathway in BE, esophagitis, and normal squamous epithelium. Western blot analysis of BMP-4, P-Smad 1/5/8, ID2, BMP RIA, BMP RII, and Smad4 expression (A) in BE and normal squamous esophagus biopsy specimens and (B) in inflamed squamous (esophagitis) and normal squamous epithelium. Results show that BMP-4 and its downstream targets P-Smad 1/5/8 and ID2 are expressed in BE and inflamed squamous epithelium, while they are not expressed in normal squamous epithelium. Smad4 and both receptors, BMP receptor IA and BMP receptor II, are expressed in BE inflamed squamous epithelium as well as normal squamous epithelium. Actin was used as a control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
BMP-4 pathway expression in normal squamous esophagus, inflamed squamous epithelium, and BE of patient biopsy specimens. H&E stainings and immunohistochemistry with fluorescein isothiocyanate (green) conjugated antibodies for BMP-4, P Smad 1/5/8, and ID2 on biopsy specimens of normal squamous epithelium, esophagitis, and BE are shown. DAPI (blue) was used as a nuclear counterstain. Immunohistochemistry shows that normal keratinizing esophageal mucosa does not show any staining for BMP-4, P-Smad, and ID2. The inflamed squamous epithelium and Barrett’s mucosa show an increased expression of BMP-4 and nuclear expression of the downstream targets including P-Smad 1/5/8, and nuclear and cytoplasmic staining for ID2, confirming activation of the BMP pathway. (Original magnification 100× and 40×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
BMP-4 pathway expression in normal squamous esophagus, inflamed squamous epithelium, and the rat BE model. The H&E stainings and immunohistochemistry using fluorescein isothiocyanate (green) conjugated antibodies for BMP-4, P-Smad 1/5/8, and ID2 of the tissue from the rat esophagus-BE model are shown. The left column shows normal nonkeratinizing squamous epithelium, inflamed squamous mucosa, and columnar mucosa resembling BE. Immunohistochemistry shows keratinizing normal rat esophageal mucosa with negative staining for BMP-4 and negative staining for the BMP-4 downstream targets. The inflamed squamous epithelium and columnar epithelium show activation of the BMP-4 pathway with increased expression of BMP-4 and nuclear expression of the downstream targets P-Smad 1/5/8 and ID2. DAPI (blue) was used as a nuclear counterstain. (Original magnification 40×.)
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
P-Smad 1/5/8 expression in cell cultures stimulated with BMP-4. (A) Western blot analysis of P-Smad 1/5/8 in primary cultured Barrett’s and squamous esophageal cells. In cultured squamous cells there is no phosphorylation of Smad 1/5/8, while cultured Barrett’s cells show high levels of P-Smad 1/5/8. Upon treatment of the squamous cell cultures with BMP-4 for 5, 10, or 20 minutes, P-Smad 1/5/8 levels are up-regulated. (B) This up-regulation in phosphorylation level of Smad 1/5/8 is effectively inhibited when the squamous cells are incubated with Noggin (a BMP antagonist). Actin was used as a control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Expression of CK7, CK10/13, and CK20 in cultured squamous cells incubated with BMP-4. Western blot analysis of CK7, CK10/13, and CK20 expression in cultured squamous cells and Barrett cells shows that in control (not treated) squamous cell cultures there is high expression of CK10/13 but CK7 or CK20 expression is absent. After 5 hours of incubation of the squamous cultures with BMP-4, CK7 and CK20 are up-regulated, while CK10/13 is still present. Actin was used as control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
CK7, CK10/13, and CK20 expression in cultured Barrett’s, nontreated squamous, and BMP-4–treated squamous cells. Immunohistochemistry with fluorescein isothiocyanate (green) conjugated antibodies shows the expression of CK7, CK10/13, and CK20 in primary cultured Barrett’s cells, squamous esophageal cells, and BMP-4–treated squamous cells. DAPI (blue) was used as a counterstain. Normal squamous cells show no expression of CK7 and CK20 but strongly express CK10/13 (left column), while Barrett cells are positive for CK7 and CK20 but not for CK10/13 (right column). After 5 days of treatment of the cultured squamous cells with BMP-4, CK7 and CK20 were up-regulated while CK10/13 expression was decreased (middle column). In the middle column (squamous cells treated with BMP-4), subpanels are included showing in detail the CK pattern change in single cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Microarray analysis comparing differentially expressed genes in squamous and BMP-4–treated squamous cells versus Barrett’s cells. (A) Scatter plots showing the comparison of microarray analysis of Barrett’s versus nontreated squamous cells and (B) Barrett’s versus BMP-4–treated squamous cells. (C) Two-dimensional dendrogram created from hierarchical clustering, according to the Pearson correlation, with the genes in a cluster displayed below each average. The length of each branch of the tree is proportional to the distance between the averages. In the upper panel, depicted in red are genes that are up-regulated in the cultured squamous cells and depicted in green are genes that are down-regulated in squamous cells. In the lower panel, depicted in red are the genes that are up-regulated in the BMP-4–treated squamous cells and depicted in green are the down-regulated genes. The expression of BE is taken as the reference (black).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions