1. The role of transforming growth factor beta-2, beta-3 in mediating apoptosis in the murine intestinal mucosa 
Nicole Dünker, Kai Schmitt, Norbert Schuster, Kerstin Krieglstein 
Gastroenterology 
Volume 122, Issue 5, Pages (May 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A–D) Expression of TGF-β2 and TGF-β3 in the (A and B) wild-type murine small intestine and (C and D) colon. TGF-β2 (arrows in A) and TGF-β3 (arrowheads in B) immunoreactive cells were found along the small intestine crypt-to-villus axis. Cells staining positively for TGF-β2 were also found in the colon (arrows in C). TGF-β3 immunoreactivity was even more abundant in the colon, where intense staining was localized in the crypts. Scale bar: A and B, 20μm; C and D, 100 μm. (E) Localization of the TβR-II in the small intestine. TβR-II is expressed throughout the intestinal mucosa, but expression seems to be more concentrated at the villus tip (white arrowheads). Scale bar: E, 25 μm. (F and G) Western blot analysis of (F) TGF-β ligand and (G) TβR expression. Homogenized wild-type mouse gut extracts (100 μg) were separated on a 12.5% sodium dodecyl sulfate–polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane. The membrane was incubated with specific antibodies for TGF-β2, TGF-β3, TβR-I, and TβR-II. Both TGF-β ligands and receptors could be detected in the gut. The level of TGF-β2 expression seems to be higher in the small intestine, whereas TGF-β3 levels are significantly higher in the colon. Expression of TβR-I and TβR-II was confirmed for the small intestine (G) and colon (data not shown).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Immunofluorescence showing the differential localization of (A) TGF-β2 and (B) TGF-β3 in small intestinal epithelial cells. TGF-β2 and TGF-β3 exhibit an isoform-specific, nonoverlapping expression pattern: TGF-β2 was mainly localized in endocrine cells (bold arrows in A), whereas TGF-β3 immunoreactivity was predominantly confined to goblet cells (arrowheads in B). Arrowheads in A demarcate goblet cells, which remained unstained for TGF-β2 immunoreactivity. Cell type identity and specificity of the labeling patterning was confirmed by (C) confocal imaging of a TGF-β2-immunoreactive cell and (D) confocal imaging of chromogranin A labeling, both visualizing basal concentration of hormone-accumulating secretory granules typical for enteroendocrine cells (small arrows in C and D). Enterocytes labeled by PBA, specifically staining mucus-secreting goblet cells (arrowheads in E), morphologically strongly resemble (B) TGF-β3–positive cells. Scale bar: A, B, and D, 20μm.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Western blot analysis of TGF-β2 and TGF-β3 expression in the small intestine of Tgfβ2+/− and Tgfβ3+/− heterozygous mice. Homogenized wild-type mouse gut extracts (200 μg) were separated on a 12.5% sodium dodecyl sulfate–polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane. The membrane was incubated with specific antibodies for TGF-β2 and TGF-β3. The level of TGF-β2 expression was found to be lower in the small intestine of Tgfβ2+/− heterozygous mice, whereas TGF-β3 levels are significantly lower in the gut of Tgfβ3+/− heterozygous mice. Densitometric values were normalized with respect to values obtained with wild-type extracts.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
TUNEL labeling of apoptotic cells in the small intestine. Apoptotic enterocytes, labeled by peroxidase-converted TUNEL staining, were mainly confined to the top of intestinal villi (arrowheads in A and arrows in C and D). Detection of DNA fragmentation (arrowheads in B) by fluorescein-dUTP incorporation (see Materials and Methods) clearly confirmed the apoptotic nature of enterocyte cell death. Many more apoptotic nuclei (arrows in C and D) were labeled in (C) wild-type mice compared with (D) Tgfβ3+/− heterozygous littermates. Scale bar: A and B, 20 μm; C and D, 100 μm.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Quantification of cell death. (A) Apoptotic levels were quantified in homogenates of small intestine (■) and colon (□) using the nucleosome assay (ELISA; see Materials and Methods). Cell death is significantly reduced in the small intestine of Tgfβ2+/− and Tgfβ3+/− heterozygous as well as in Tgfβ2+/− Tgfβ3+/− double heterozygous mice compared with wild-type littermates. In the colon, however, apoptotic levels seem to be unaffected in Tgfβ2+/−, Tgfβ3+/−, and Tgfβ2+/− Tgfβ3+/− mice. (B) Apoptotic levels were quantified in small intestinal tube sections using the TUNEL assay. The number of apoptotic cells is clearly reduced in Tgfβ2+/− and Tgfβ3+/− heterozygous as well as in Tgfβ2+/− Tgfβ3+/− double heterozygous mice compared with wild-type littermates. Values are means ± SEM. *P < 0.01; **P < 0.005; unpaired Student t test.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Quantification of (A) cell proliferation, (B) cell diameter, and (C) villus length. Neither cell proliferation rate as detected by (A) PCNA stain (see Materials and Methods) nor (B) endothelial cell diameter is altered in the small intestine of Tgfβ2+/− and Tgfβ3+/− heterozygous as well as Tgfβ2+/− Tgfβ3+/− double heterozygous mice compared with wild-type littermates. (C) Small intestinal villi of Tgfβ2+/− and Tgfβ3+/− heterozygous mice exhibit a significant increase in length compared with wild-type villi. Values are means ± SEM. **P < 0.005; unpaired Student t test.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
(A and B) Analysis and (C and D) densitometric quantification of Bcl-xL and Bcl-2 expression levels in the small intestine of wild-type and Tgfβ2+/− and Tgfβ3+/− heterozygous mice by Western blotting analysis using (A) Bcl-xL– and (B) Bcl-2–specific antibodies. Protein levels of both Bcl-xL and Bcl-2 are significantly increased in Tgfβ2+/− and Tgfβ3+/− heterozygous compared with wild-type mice. *P < 0.001; **P < 0.005; unpaired Student t test.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions