1. Volume 141, Issue 3, Pages 1102-1113.e8 (September 2011)
Interferon Regulatory Factor-2 Regulates Exocytosis Mechanisms Mediated by SNAREs in Pancreatic Acinar Cells 
Hirosato Mashima, Taku Sato, Yasuo Horie, Yuko Nakagawa, Itaru Kojima, Toshiaki Ohteki, Hirohide Ohnishi 
Gastroenterology 
Volume 141, Issue 3, Pages e8 (September 2011)
DOI: /j.gastro
Copyright © 2011 AGA Institute Terms and Conditions

2. Figure 1
Morphologic studies of pancreatic acinar cells in Irf2+/+, Irf2+/−, and Irf2−/− mice. (A) Gross appearance, H&E staining (scale bars, 50 μm), and EMs (scale bars, 10 μm [original magnification, ×300], 2 μm [original magnification, 2000× and 4000×]) are shown. The panels with higher magnification of the boxed areas are indicated by arrows. Fused ZGs with the apical membrane (arrowhead), secreted contents of ZGs (asterisks), and apical ductal area filled with secreted contents (Ω) were observed in Irf2+/+ and Irf2+/− acini, whereas they could not be detected in Irf2−/− acini. (B) EMs of the subapical area of Irf2−/− acini (scale bar, 10 μm). Closely located granules and kissing granules were observed. (C) An EM analysis of ZG abundance (i), size (ii), fusion (iii–v), and the apical lumen area (vi). For each strain, 300 cells from at least 3 mice were quantified. Data are expressed as means ± SD. *P < .05, **P < .01. Data are also listed in Supplementary Table 2.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
Alterations of amylase staining and the regulated exocytosis in Irf2−/− acini. (A) Immunofluoresence staining of amylase (red) in Irf2+/+, Irf2+/−, and Irf2−/− pancreas (scale bars, 25 μm). The subluminal actin was stained with fluorescein isothiocyanate-conjugated phalloidin, and the nuclei were stained with 4′,6-diamidino-2-phenylindole. (B) Western blotting of amylase in the homogenate of pancreas. α-Tubulin was used as an internal control. Right panel shows the ImageJ densitometry analysis (ImageJ software; Image J, National Institutes of Health, Bethesda, MD). Results are expressed as the means ± standard deviation. (C) Amylase secretion from dispersed pancreatic acini in Irf2+/+, Irf2+/−, and Irf2−/− mice. Dispersed acini were incubated in 0 pmol/L, 10 pmol/L, and 100 pmol/L CCK-8 for 30 minutes. Amylase release into the supernatant was quantified and expressed as a percentage of the total amylase in the acini at the beginning of the incubation. Data are expressed as the means ± standard deviation of triplicate reactions and are representative of 2 independent experiments with similar results. *P < .05, **P < .01.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
Alterations of SNARE proteins in Irf2−/− pancreas. (A) Total cellular homogenates of pancreas from 2-month, 3-month, and 5-month old Irf2+/− and Irf2−/− mice were prepared. Western blotting was carried out, and α-tubulin was used as an internal control. The blots are the representative of 2 independent experiments with similar results. ImageJ densitometry analyses are shown in Supplementary Figure 2. (B) The localization of SNARE and SM proteins was demonstrated by immunohistochemistry. The nuclei were stained with 4′,6-diamidino-2-phenylindole, and subluminal actin was stained with fluorescein isothiocyanate-conjugated phalloidin (scale bars, 25 μm).
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Figure 4
Autophagy was activated in Irf2−/− acini at basal state (A) EMs showed a number of autophagic vacuoles (arrowheads) in Irf2−/− acini. High-density materials in vacuoles are indicated by asterisks. (B) Autophagic vacuoles containing membrane whorls are indicated by arrows. (C) Autophagic cell death was detected in some pancreatic acinar cells showing ruptured plasma membrane with multiple vacuoles in the cytoplasm and the absence of chromatin condensation in the nucleus (scale bars, 10 μm). (D) The expression level of LC3-II was observed to increase in Irf2−/− pancreas. (E) Active trypsin was measured in whole cell lysates by a fluorimetric assay. Values are the means ± standard deviation from n = 5–6 for each strain. *P < .05.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. Figure 5
Morphometric studies of the salivary glands and pancreatic islets and the expression of SNARE and SM proteins in parotid gland in Irf2+/− and Irf2−/− mice. (A) H&E staining of submandibular gland, parotid gland, and pancreatic islet in Irf2+/− and Irf2−/− mice is shown. Semithin sections of salivary glands were stained with toluidine blue. Immunohistochemical staining for insulin and glucagon using serial sections is also shown. Scale bars, 200 μm (H&E), 20 μm (toluidine blue) (B) Total cellular homogenate of parotid gland from 3-month-old mice was prepared for Western blotting. (C) H&E staining and immunohistochemical staining for SNAP23 using serial sections of parotid gland are shown (Scale bars, 50 μm).
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. Figure 6
The alteration of the regulated exocytosis in AR42J cells expressing full-length or dominant-negative IRF2. (A) Total cellular homogenates of GFPcont, GFPirf2, and GFPirf2dn AR42J cells were prepared for Western blotting. (B) Reverse-transcription polymerase chain reaction for full-length and dominant-negative Irf2 was performed. The right end lane is 100-base pair ladder size marker, and upper panel shows the locations of the primers designed The expression of β-actin was used as an internal control. (C) The cells were incubated at 37°C for 15 minutes in the presence or absence of 100 pmol/L CCK-8, 5 μmol/L A23187, and/or 500 μmol/L 8Br-adenosine 3′,5′-cyclic monophosphate. Amylase secretion and contents were measured, and amylase release was expressed as a percentage of total amylase in AR42J cells at the beginning of the incubation. Data represent the means ± SD (n = 4) and are representative of 3 independent experiments with similar results. *P < .05, **P < .01.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

8. Figure 7
The expression of IRF2 and the alterations of Irf2+/− and Irf2−/− pancreas in supramaximal cerulein-induced pancreatitis. (A) Cerulein pancreatitis was induced in wild-type mice (50 μg/kg/h intraperitoneally, 5 times, n = 3, [Ce]) and the control group received similar injections of normal saline (n= 3, [Ns]). Western blotting was carried out for IRF2 and α-tubulin. The lower panel shows the ImageJ densitometry analysis. Values are the means ± standard deviation (SD). *P = (B) Cerulein pancreatitis was induced in Irf2+/− and Irf2−/− mice (50 μg/kg/h intraperitoneally, 12 times, n = 2–3, [Ce]; normal saline, [Ns]), and H&E staining is shown. Scale bars, 500 μm, 100 μm (higher magnification). The experiments were repeated 3 times independently, and the summary of histologic scores is listed in Supplementary Table 3. (C) The serum digestive enzyme levels. Values are the means ± SD of n = 6–7 for each group. *: P < .05. (D) Active trypsin level in whole cell lysates. Values are means ± SD from n = 6–7 for each group. *P < .05. (E) The expression of LC3. The blots are representative of 3 independent experiments with similar results. (F) Immunohistochemistry of Munc18c in cerulein-induced pancreas. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Upper panels show the phase contrast images of the same area. Scale bars, 50 μm.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

9. Supplementary Figure 1
The expression of IRF2 and IFNAR1 in the pancreas and salivary gland. (A) The expression of IRF2 in the pancreas and salivary gland was studied in Irf2+/+, Irf2+/−, and Irf2−/− mice by Western blotting. (B) The expression of Irf2 and Ifnar1 was studied in the total, exocrine and endocrine pancreas of Irf2+/−, Irf2−/−, and Irf2−/−Ifnar1−/− mice by RT-PCR. In the panel of Irf2 (264 base pair), lower band (164 base pair) represents the splicing variant, which lacks the third exon, and a nonsense protein will be produced by a frame shift.6
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

10. Supplementary Figure 2
Summary of the ImageJ densitometry analyses of soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Sec1/Munc18 protein levels shown in Figure 3A. The data came from blots of 2 independent experiments. Data represent the means ± standard deviation. *P < .05, **P < .01 compared with Irf2+/− pancreas. SM, Sec1/Munc18; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

11. Supplementary Figure 3
The abnormalities in Irf2−/− acini were not rescued by disabling type I IFN signaling. (A) The gross appearance of the pancreas from Irf2−/−Ifnar1−/− mice. (B) EMs of Irf2−/−Ifnar1−/− acini (scale bars, 10 μm). The panels with higher magnification of the boxed area are indicated by arrows. The spread of ZGs throughout the cytoplasm was not rescued in Irf2−/−Ifnar1−/− acini, suggesting that this abnormality was independent of type I IFN signaling. Also note that neither granules fused with the apical membrane nor secreted contents into the apical lumen were found in Irf2−/−Ifnar1−/− acini. The analysis of EM images of Irf2−/−Ifnar1−/− acini is listed in Supplementary Table 2. EM, electron micrograph; IFN, interferon; ZG, zymogen granules.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

12. Supplementary Figure 4
Summary of messenger RNA levels in the total, exocrine, and endocrine pancreas from Irf2+/− and Irf2−/− mice. Relative expression of SNARE and SM genes were measured by quantitative real-time PCR. To discriminate the changes of expression precisely, we also selected acinus and islet and compared the messenger RNA level. To evaluate the contamination of islet into the samples from acinus, we measured the expression of Snap25, insulin (I, II), and proglucagon. Data represent the means ± standard deviation of triplicate reactions from 2 independent experiments. **P < .01. SM, Sec1/Munc18; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

13. Supplementary Figure 5
The expression of junctional markers in Irf2+/− and Irf2−/− pancreas. (A) Immunofluoresence study (Nuclei were stained with DAPI; scale bars, 50 μm), and (B) Western blotting of junctional markers were done to examine their expression. The panels with higher magnification of the boxed area are indicated by arrows. The expression of junctional markers was markedly reduced in Irf2−/− pancreas. DAPI, 4′,6-diamidino-2-phenylindole.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

14. Supplementary Figure 6
Western blotting of SNARE and SM proteins in GFPcont, GFPirf2 (full-length IRF2 over-expressing), and GFPirf2dn (dominant-negative IRF2 over-expressing) AR42J cells. Summary of the ImageJ densitometry analyses is shown in the lower panel. The data came from blots of 3 independent experiments. Values are the means ± standard deviation. SM, Sec1/Munc18; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.
Gastroenterology  , e8DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions