1. Volume 134, Issue 2, Pages 544-555.e3 (February 2008)
Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis 
Jens T. Siveke, Clara Lubeseder–Martellato, Marcel Lee, Pawel K. Mazur, Hassan Nakhai, Freddy Radtke, Roland M. Schmid 
Gastroenterology 
Volume 134, Issue 2, Pages e3 (February 2008)
DOI: /j.gastro
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2. Figure 1
Effect of the γ-secretase inhibitor DBZ on the pancreas. (A) DBZ treatment alters pancreas morphology as shown by H&E staining. (Inset) DBZ-induced conversion of intestinal crypt cells into goblet cells as shown by periodic acid–Schiff staining. Scale bar = 50 μm. (B and C) Rate of BrdU-positive and cleaved caspase-3–positive acinar cells in DBZ- versus vehicle-treated mice (P = .184 for BrdU; P = for cleaved caspase-3). (D) Reduced pancreas/body weight index in DBZ-treated mice (**P = .002). (E) Real-time qRT-PCR analysis of Notch signaling members, Notch target genes, and exocrine genes (*P < .05, **P < .01). (F) Image intensity display of expression levels of genes commonly activated during acute pancreatitis and of genes involved in Notch signaling.
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3. Figure 2
Analysis of exocrine cell differentiation in pancreata after DBZ treatment. (A) Western blot analysis of amylase, β-catenin, and actin (loading control) expression in DBZ- or vehicle-treated pancreatic whole cell lysates. (B and C) Immunofluorescence staining for β-catenin (green) and double immunofluorescence staining for amylase (green) and β-catenin (B, red) or E-cadherin (C, red) shows cytoplasmic localization of β-catenin in DBZ-treated acini. Arrowheads highlight cytoplasmic expression. (D) Immunofluorescence staining for clusterin shows higher expression levels in DBZ-treated acinar structures. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue); i = islet; scale bar = 50 μm.
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4. Figure 3
Cerulein-induced acute pancreatitis in DBZ- or vehicle-treated mice. (A) Notch1-GFP mice reveal increased expression of Notch1 on d3 postinduction of pancreatitis. Arrowhead indicates Notch1-positive acinar cells. (B) Experimental setup for the induction of cerulein-induced pancreatitis. Mice were injected with BrdU 2 hours before they were killed (s). (C) H&E staining at d1 pancreatitis shows acute tissue reaction of both vehicle- and DBZ-treated mice; insets show magnification with infiltrating cells between acini. (D) H&E staining at d3 pancreatitis shows impaired pancreas regeneration in mice treated with the γ-secretase inhibitor DBZ. Insets show magnifications with details of the regenerating acinar structures. (E) Morphometric assessment of acini shows a significant reduction in DBZ-treated mice at d3. (F) BrdU-positive acinar cells in DBZ- and vehicle-treated mice. Black bars, d1; gray bars, d3. At d3, DBZ induces a significant reduction of acinar cell proliferation (*P = .032). (G) Significant increase in cleaved caspase-3–positive acinar cells in DBZ-treated mice 3 days after pancreatitis (**P = .006). Scale bar = 50 μm.
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5. Figure 4
Effect of DBZ treatment in d3 pancreatitis. (A) H&E staining shows a pancreatic section containing an area with impaired tissue regeneration and neighboring regenerated acini. Immunohistochemical staining for CD45 and F4/80 shows infiltrating leukocytes and macrophages. Immunostaining for amylase, PDX1, and clusterin suggests that amylase-negative cells are immature acinar cells. Insets show higher magnification. (B) Double immunofluorescence staining for amylase (green) and E-cadherin or β-catenin (red) reveals higher and partially cytoplasmic expression (arrowheads) in DBZ-treated acinar cells. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue); i = islet; scale bar = 50 μm.
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6. Figure 5
Characterization of Notch1-deficient exocrine pancreata. (A) X-gal staining shows Cre-induced recombination in all pancreatic compartments in 7-week-old N1KO mice. (B) Real-time qRT-PCR of Notch1 mRNA in pancreata of Ptf1a+/+, Notch1f/f, and Ptf1a+/Cre(ex1); Notch1f/f mice. (C) Western blot analysis shows absence of Notch1-IC in N1KO pancreatic whole cell lysates. (D) Double immunofluorescence staining for amylase (green) and β-catenin (red) in N1WT and N1KO mice. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue); scale bar = 50 μm.
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7. Figure 6
d3 pancreatitis in N1WT and N1KO mice. (A) H&E staining shows impaired tissue regeneration in N1KO pancreata. Insets show an area with regenerated acini. Immunohistochemistry for amylase shows fully differentiated acini in N1WT pancreas and many acinar cells with weak amylase staining in N1KO pancreas. (B) Morphometric assessment of acini shows a significant reduction in N1KO mice at d3. (C) BrdU-positive acinar cells are significantly increased in N1KO mice (**P = .0024). (D) Analysis of cleaved caspase-3–positive cells shows significantly more acinar cell apoptosis in N1KO mice (**P = .0016). (E) PDX1 and clusterin immunohistochemistry in N1KO mice shows expression in exocrine pancreatic cells. (F) Increased expression of β-catenin in N1KO compared with N1WT mice. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (blue); scale bar = 50 μm. (G) Western blot analysis reveals increased expression of β-catenin in N1KO mice.
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8. Figure 7
Notch and β-catenin signaling in acinar cells. (A) Amylase protein expression in pancreatic whole cell lysates and different cell lines reveals expression in cells demonstrating exocrine cell characteristics. (B) Analysis of Notch1-IC protein expression and inhibition in cells. Notch1-IC is expressed in cells and can be inhibited by treatment with γ-secretase inhibitors L685,458 and DAPT for 48 hours. (C) Treatment of cells with γ-secretase inhibitors L685,458 and DAPT for 48 hours leads to down-regulation of Hes1 mRNA. (D) Luciferase activity as measured by β-catenin/TCF TOP/FOP-FLASH ratio shows increased activity when transfected with Wnt1 or constitutively active S33 β-catenin for 48 hours in cells. (E) Dose dependency of TOP activity by different amounts of S33 β-catenin. TOP and indicated amounts of S33 β-catenin were cotransfected and luciferase activity was measured after 48 hours. Results are the mean ± SD of triplicates and are representative of at least 3 independent experiments.
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9. Figure 8
Notch1 regulates β-catenin–dependent transcriptional activity in cells. (A) S33 β-catenin–induced TOP activity is inhibited in a dose-dependent manner by cotransfecting with either Notch1-IC or Notch1ΔE. (B) Inhibition of S33 β-catenin–induced TOP activity by Notch1ΔE can be modulated by DAPT treatment cells were treated with indicated amounts of DAPT. (C) Treatment of cells with siRNA against either Notch1 or Notch2 leads to specific down-regulation of the respective Notch receptor. (D) RBP-Jκ–dependent transcriptional activity is suppressed after siRNA-mediated knockdown of Notch1. (E) S33 β-catenin–induced TOP activity is increased by siRNA-mediated knockdown of Notch1 using 2 different Notch1-specific siRNAs. (F) Notch1-IC is a stronger inhibitor of S33 β-catenin–induced TOP activity than Notch1-ICΔRBP, demonstrating the importance of a functional RAM domain. Results are the mean ± SD of triplicates measured at 48 or 72 hours for siRNA experiments and are representative of at least 4 independent experiments.
Gastroenterology  , e3DOI: ( /j.gastro )
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