1. Volume 145, Issue 3, Pages 625-635 (September 2013)
AP-1B−Mediated Protein Sorting Regulates Polarity and Proliferation of Intestinal Epithelial Cells in Mice 
Koji Hase, Fubito Nakatsu, Masumi Ohmae, Kazushi Sugihara, Noriko Shioda, Daisuke Takahashi, Yuuki Obata, Yukihiro Furusawa, Yumiko Fujimura, Taisuke Yamashita, Shinji Fukuda, Hiroshi Okamoto, Masahide Asano, Shigenobu Yonemura, Hiroshi Ohno 
Gastroenterology 
Volume 145, Issue 3, Pages (September 2013)
DOI: /j.gastro
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2. Figure 1
Increased mortality and enlarged small intestine in Ap1m2-/- mice. (A) Kaplan–Meier survival analysis of Ap1m2+/- (solid line, N = 164) and Ap1m2-/- mice (dotted line, N = 114) mice. A statistically significant difference was observed between the 2 groups (P < .001). (B) Growth curve of Ap1m2+/- mice (square, N = 5 and 7 for female and male mice, respectively) and Ap1m2-/- mice (circle, N = 3 and 4 for female and male mice, respectively). Blank and filled symbols represent female and male mice, respectively. (C [arrows indicate the duodenum] and D) Macroscopic views of the gastrointestinal tract of Ap1m2+/- and Ap1m2-/- mice. (E) Higher magnifications of the dissected duodenum from Ap1m2+/- and Ap1m2-/- mice. Scale bars, 2 mm. (F) Weight of the small intestine was measured around weaning (age, 3–5 wk; N = 12 and 7 for +/- and -/-, respectively) and in adults (age 7–10 wk, N = 12). The value is normalized to body weight. Data are presented as the mean ± SD. **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

3. Figure 2
Epithelial hyperplasia caused by enhanced cell proliferation in the small intestine of Ap1m2-/- mice. (A) Duodenal tissue sections were stained with H&E for histologic analysis. Lower panels are magnified images of the crypt region. (B) The length of small intestinal crypts and villi was measured by microscopy. Data are presented as the mean ± SD (N = 6 and 4 for +/- and -/-, respectively). **P < .01. (C) Scanning electron microscopic analysis of tissue morphology of the upper small intestine. (D) Tissue sections prepared from the upper small intestine were stained with anti-Ki67 antibody (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Arrows indicate the crypt regions. Scale bars: (A) 200, (C, upper, and D) 100, and (C, lower) 20 μm. Data are representatives of (C) 2 or (A and D) 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

4. Figure 3
Impairment in cell polarity formation in the small intestine of Ap1m2-/- mice. (A) Tissue sections of the upper small intestine were stained with antibodies specific for markers of tight junctions (ZO-1), and basolateral (Na+, K+–adenosine triphosphatase [ATPase], LDLR) and basal (β4-integrin) plasma membranes. Solid lines and dotted lines in the top panels indicate apical and basal plasma membranes, respectively. Arrows indicate localization of LDLR in cytoplasmic vesicles. (B) Subcellular localization of EphB2 was examined in the crypt of the upper small intestine. Arrowheads represent apical localization of EphB2. (C) Subcellular localization of apical marker proteins (sucrase and villin) was examined. The arrowhead represents cytoplasmic vesicular staining of sucrase. Scale bars, 10 (LDLR, sucrase), 15 (EphB2), 20 (ZO-1, Na+, K+-ATPase, villin), and 200 (ß4-integrin) μm. Representative images from (A and C) 3 or (B) 5 independent experiments are shown. DAPI, 4′,6-diamidino-2-phenylindole.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

5. Figure 4
Transmission electron microscopic analysis of the cell structure of small intestinal epithelial cells. (A) Arrows indicate ectopic microvillus formation on the lateral plasma membrane. Arrowheads indicate enlarged lysosome-like vacuoles. Representatives from 3 independent experiments are shown. (B) The regions around the Golgi apparatus are shown. The right panels are higher magnification images of the trans-Golgi network region depicted with a square in the left panels. Arrowheads indicate endosome-like vesicular structures. Representative images from 3 independent experiments are shown. Scale bars, 1 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

6. Figure 5
Serum profiles of Ap1m2+/- and AP1m2-/- mice. Serum samples from 8- to 10-week-old mice were analyzed for the indicated biochemical parameters. Data are mean ± SD (N = 4). *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

7. Figure 6
Cytoplasmic accumulation of E-cadherin associated with an increase in the nuclear translocation of β-catenin in intestinal epithelium of Ap1m2-/- mice. (A) Immunofluorescent analysis of E-cadherin (green) in the intestinal villous epithelium. Nuclei are stained with 4′,6-diamidino-2-phenylindole. Arrows indicate E-cadherin accumulation in the cytoplasmic granules in Ap1m2-/- cells. Scale bars, 10 μm. (B) Immunoblot analysis of E-cadherin in whole-cell lysate and the cytoplasmic fraction prepared from duodenal epithelium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also was detected as an internal control. (C) Immmunoblot analysis of β-catenin in whole-cell lysate and immunoprecipitated fractions using anti–E-cadherin antibody or control IgG. (D) Immunohistochemical detection of β-catenin in formalin-fixed, paraffin-embedded sections. The lower panels are the magnifications of the rectangles in the upper panels. Scale bars, 200 μm. Arrows represent nuclear localization of β-catenin. (E) Immunoblot analysis of β-catenin in the nuclear fraction prepared from duodenal epithelium. The nuclear protein histone H3 also was detected as an internal control. (B, C, and E) The band intensity was measured by densitometry. Data are mean ± SD of 3 or 4 different mice per group. **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

8. Figure 7
β-catenin signaling is enhanced in the intestinal epithelium of Ap1m2-/- mice, leading to excessive cell proliferation. (A) Microarray analysis of gene expression of β-catenin/Tcf4 target molecules and cargo molecules for the AP-1B complex manifested up-regulation of these molecules in Ap1m2-/- cells. Pooled data of 3 individual samples obtained from duodenal epithelium of different mice are shown. (B) The duodenal tissue sections were stained for β-catenin/TCF target molecules, CD44 and c-Myc. Note that the cells in intestinal lamina propria are also positive for CD44. The representatives of 3 independent experiments are shown. (C) Duodenal sections of Ap1m2-/- mice that received an intraperitoneal injection of IWR-1 or vehicle were stained for H&E, CD44, and Ki67. The number of Ki67-positive cells per the crypt-villous unit was quantified. Data are mean ± SD of 4 different mice per group. **P < .01. Scale bars: (B) 40 or (C) 200 μm. DAPI, 4′,6-diamidino-2-phenylindole.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions