1. Volume 134, Issue 5, Pages 1544-1554 (May 2008)
Cell Adhesion Molecule 1 Is a Novel Pancreatic–Islet Cell Adhesion Molecule That Mediates Nerve–Islet Cell Interactions 
Yu–Ichiro Koma, Tadahide Furuno, Man Hagiyama, Kazuyuki Hamaguchi, Mamoru Nakanishi, Mari Masuda, Seiichi Hirota, Hiroshi Yokozaki, Akihiko Ito 
Gastroenterology 
Volume 134, Issue 5, Pages (May 2008)
DOI: /j.gastro
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2. Figure 1
Expression of CADM1 in the mouse pancreas. (A) Protein samples were prepared from pancreatic tissues of mice at the indicated ages (in days) and from adult mouse liver (positive control), pretreated with PNGase F (+) or not treated (-), and blotted with anti-CADM1. After stripping, blots were reprobed with an anti–β-actin antibody to indicate the protein loading per lane. E, embryonic day. (B) Sections of the pancreas at E11 (a), E14 (b), E16 (c), E18 (d), postnatal day 2 (e), and 4 weeks of age (f and g) were incubated with the anti-CADM1 (rabbit polyclonal) antibody and stained with aminoethylcarbazole. The nuclei were counterstained with hematoxylin. In a, p and s indicate the primordia of the pancreas and stomach, respectively. In d and e, arrowheads outline the developing islets; black arrowheads indicate CADM1-positive islet cells. In f and g, arrows and asterisks indicate CADM1-positive nerves and islet cells, respectively. Original magnification: a and b, 300×; c–e, 400×; f, 200×; g, 1000×. (C) Islets and exocrine acini were isolated separately from mouse pancreas by enzymatic homogenization. Isolations were confirmed morphologically in H&E-stained samples (a). Protein lysates and total RNA were prepared from the isolated tissues, and were subjected to Western blot (b) and reverse-transcription polymerase chain reaction (c) analyses, respectively. Blots were probed with anti-CADM1 or anti–α-amylase, and a silver-stained gel shows the total protein per lane (b). Complementary DNAs generated from total RNA were polymerase chain reaction–amplified with a primer set specific for either CADM1 or glyceraldehyde-3-phosphate dehydrogenase (c). (D) Sections of the pancreas at E18 (a), 1 week (b and c), and 4 weeks (d) were double-labeled with antibodies against CADM1 (3E1) and PGP9.5, and stained with Cy3- and Cy2-conjugated antibodies, respectively. The yellow area (depicted by arrows in b–d) indicates colocalization of the signals. The boxed area is enlarged in the inset in d. In a, b, and d, g indicates the intrapancreatic ganglion. Original magnification: a–c, 600×; d, 800×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
Double-staining immunofluorescence of (A) murine and (B) human adult pancreases. Sections were incubated with antibodies against CADM1 (3E1; a, d, g, and j) and islet hormones (insulin, b; glucagon, e; somatostatin, h; and PP, k), and stained with Cy3- and Cy2-conjugated antibodies, respectively. Both images are merged in c, f, i, and l. In A, d, arrowheads indicate CADM1 signals, suggesting that CADM1 may mediate heterotypic contacts between α cells and other cell types. Original magnification: 400×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

4. Figure 3
Involvement of CADM1 in aggregation of αTC6 cells. (A) Western blot analysis of CADM1 in αTC6 cells. Protein samples were prepared from the parent αTC6 cells, cells transfected with an siRNA vector containing either CADM1-targeting (αTC6siRNA-CADM1) or scrambled (αTC6siRNA-scrambled) sequences, mouse neonate SCG, and adult lungs (positive control).5 Blots were probed with anti-CADM1. After stripping, blots were reprobed with an anti–β-actin antibody to indicate the protein loading per lane. (B) Double-staining immunofluorescence of αTC6 cells. Cells were cultured on glass-bottomed dishes, incubated with antibodies against CADM1 (3E1; b) and glucagon (c), and stained with Cy3- and Cy2-conjugated antibodies, respectively. Merged images (d). Differential interference contrast image (a). Original magnification: 400×. (C) Cell aggregation assay. Single-cell suspensions of αTC6 cells were preincubated with 9D2 or U04 at the indicated concentrations, and then were rotated for 1 hour. The percentages of cells incorporated in aggregates were calculated, and the data are presented as mean ± SE of triplicate samples (a). *P < .05 by t test when compared with values obtained in the absence of antibodies. Representative phase-contrast and CADM1 immunofluorescence (insets) images of the aggregates are shown (b–d): b, no antibody; c, 9D2 (14 μg/mL); d, U04 (14 μg/mL).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Coculture of αTC6 cells with SCG neurons. (A) Representative micrographs of the αTC6–SCG cocultures. After coculturing αTC6 cells on SCG neurite networks for 3 hours, the coculture was incubated with anti-CADM1 and antiglucagon, and stained with Cy3-conjugated (b) and Cy2-conjugated (c) antibodies. Merged images (d). Differential interference contrast image (a). Original magnification: 1000×. (B) Involvement of CADM1 in the attachment of αTC6 cells to SCG neurites. The coculture of αTC6 cells on SCG neurite networks was developed in the presence of 9D2 or U04. The concentration at which each antibody was added to the coculture is shown. After 3 hours of coculture, the number of αTC6 cells attached to SCG neurites sprouting from 1 neuron was calculated, and the means ± SE of triplicate samples are shown. *P < .05 by t test when compared with values obtained in the absence of antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

6. Figure 5
Response of αTC6 cells to SV-evoked nerve activation. (A–E) Ca2+ mobilization in SCG neurites and associated αTC6 cells after SV addition into (A and B) 2-dimensional and (C and D) 3-dimensional cocultures as indicated by Fluo-3 or Fluo-8 fluorescence. Representative results are shown. After 2 days in coculture, αTC6 cells on SCG neurite networks were loaded with Fluo-3 or Fluo-8, and were stimulated with SV (arrows; t = 0). Subsequently, Fluo-3 or Fluo-8 fluorescence was measured every (B) 1 or (A and C–E) 3 seconds. Differential interference contrast (dic) and fluorescence images from the indicated times are shown in panels above the graphs. In the t = 0 panels, nerves are outlined by dotted lines, and, in the next panels, several regions of interest (boxed by white lines) are defined in the center of αTC6 cells (square) and nearby segments of SCG neurites (rectangle). The fluorescence intensity is displayed in pseudocolor, with blue indicating the least fluorescence. The scale is shown at the right of each panel. Traces of fluorescence intensity for each region of interest are shown as line graphs. (D) Red asterisks depict out-of-focus cells. (F) Involvement of CADM1 in the response of αTC6 cells to nerve activation. αTC6 cells with an increase of more than 25 arbitrary units during the trace period were considered to be responding to nerve activation in 2-dimensional cocultures. The fraction of responding αTC6 cells among all αTC6 cells associated with neurites was calculated for each experimental group. The means ± SE of triplicate dishes per group are shown. *P < .01 by t test when compared with control αTC6 cells.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Immunohistochemical staining of CADM1 in ICTs. (A) Representative results of CADM1-positive cases. In a–c, sections from human normal pancreas (a) and ICTs (b, case 10; c, case 2) were incubated with the anti-CADM1 (rabbit polyclonal) antibody and stained with aminoethylcarbazole. The nuclei were counterstained with hematoxylin. In d–f, sections from ICTs (d, case 18; e, case 10; and f, case 2) were double-stained with antibodies against CADM1 and islet hormones (d, gastrin; e, insulin; and f, glucagon), and stained with Cy3- and Cy2-conjugated antibodies, respectively. Original magnification: a, 150×; b and c, 300×; and d–f, 200×. (B) Association of ICTs with nerves. In a and b, sections from normal human pancreas (a) and a human ICT (b, case 13) were incubated with anti-PGP9.5 and stained with aminoethylcarbazole. The nuclei were counterstained with hematoxylin. Boxed areas are enlarged in the insets.19 In c–f, sections from ICTs (c and d, case 5; e and f, case 10) were double-stained with antibodies against CADM1 (3E1) and S-100 protein, and stained with Cy3- and Cy2-conjugated antibodies, respectively. Boxed areas in c and e are enlarged in d and f, respectively. Arrows in d and f indicate direct contacts between CADM1-positive nerves and ICT cells. Islet cells were weakly immunoreactive for PGP9.5, as reported previously.19f, nerve funiculus; g, intrapancreatic ganglion; i, islet. Original magnification: a and b, 150×; c and e, 400×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions