1. Volume 137, Issue 1, Pages 350-360.e5 (July 2009)
Impaired Autolysosome Formation Correlates With Lamp-2 Depletion: Role of Apoptosis, Autophagy, and Necrosis in Pancreatitis 
Franco Fortunato, Heinrich Bürgers, Frank Bergmann, Peter Rieger, Markus W. Büchler, Guido Kroemer, Jens Werner 
Gastroenterology 
Volume 137, Issue 1, Pages e5 (July 2009)
DOI: /j.gastro
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2. Figure 1
Evaluation of pancreatic necrosis after chronic alcohol exposure and endotoxemia. (A) Representative image of TUNEL+ nuclei according to their apoptosis- or necrosis-like morphology. Arrowhead indicates TUNEL+ nuclei with condensed chromatin (apoptosis) and arrows more diffuse chromatin distribution (necrosis). (B) TUNEL+ positivity was evaluated in 20 fields for apoptosis- and necrosis-like appearance. Plotted were means ± SEM for 4 animals per group. *P = vs pair-fed rats 24 hours after LPS (necrosis); **P = vs alcohol-fed rats 24 hours after LPS (apoptosis); ***P = vs alcohol-fed rats 24 hours after LPS (necrosis); ****P = vs alcohol-fed 24 hours after LPS (necrosis); P = .511 vs alcohol-fed rats 24 hours after LPS (necrosis). (C) Representative HMGB1 immunoblot obtained after separation of tissue into nuclear (“N”) and cytosolic (“C”) fractions. (D) Representative HMGB1 immunoblot obtained from whole pancreatic homogenate. The normalized ratios were plotted as means ± SEM for 4 animals per group. *P = vs alcohol-fed rats 3 hours after LPS injection; #P = vs alcohol-fed rats 3 hours after LPS injection.
Gastroenterology  , e5DOI: ( /j.gastro )
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3. Figure 2
Transmission electron microscopy of mitochondria in pancreatic acinar cells after ethanol and endotoxemia. (A) Representative EM image of pancreatic acinar cells. Arrows indicate damaged mitochondria with signs of pathologic changes such as calcification of the mitochondrial membrane, mitochondrial lysis, or injured criste (see also Supplementary Figure 2). (B) Quantitation of ultrastructural changes in mitochondria. Plotted as means ± SEM of 4 individuals per group. *P < vs pair-fed control rats; **P < vs pair-fed control; and ***P = vs alcohol-fed rats. (C) Quantitation of normalized ATP concentrations per total protein concentration. Plotted as means ± SEM of 4 or 5 individuals per group. *P = vs alcohol plus LPS; #P = vs LPS. (D) Quantitation of the ADP/ATP ratio in the alcohol and pair-fed animals 24 hours after LPS. Plotted as means ± SEM of 5 individuals per group. *P = vs alcohol-fed rats 24 hours after LPS injection.
Gastroenterology  , e5DOI: ( /j.gastro )
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4. Figure 3
Evaluation of pancreatic caspase activities and proteolytic maturation after chronic alcohol feeding and endotoxemia. (A) Quantitation of caspase-3 activity plotted as means ± SEM of 5 animals per group. *P < vs pair-fed animals 24 hours after LPS; **P = vs alcohol-fed animals; ***P = vs alcohol-fed animals 24 hours after LPS. (B) Representative immunoblot of pro- and active caspase-3. (C) Quantitation of caspase-9 activity plotted as means ± SEM of 5 animals per group. *P = vs alcohol-fed rats; **P = vs alcohol-fed rats 24 after LPS. (D) Representative immunoblot of pro- and active caspase-9.
Gastroenterology  , e5DOI: ( /j.gastro )
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5. Figure 4
Determination of pancreatic cathepsin B and trypsin activities after chronic alcohol feeding and endotoxemia. (A) Quantitation of cathepsin B activity was plotted as means ± SEM of 5 animals per group. *P = vs pair-fed rats 24 hours after LPS; **P = vs alcohol-fed rats; ***P < vs alcohol-fed rats 24 hours after LPS; ****P = vs alcohol-fed rats 24 hours after LPS. (B) Quantitation of trypsin activity was plotted as means ± SEM of 5 animals per group. *P < vs pair-fed rats 24 hours after LPS; **P = vs alcohol-fed rats; ***P < vs alcohol-fed rats 24 hours after LPS; ****P = vs alcohol-fed rats 24 hours after LPS.
Gastroenterology  , e5DOI: ( /j.gastro )
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6. Figure 5
Electron microscopy assessment of autophagosomal and autolysosomal formation. (A) Representative EM image of pancreatic acinar cells. The arrow indicates a typical autophagosomes containing mitochondria and other intracellular components. (B) Quantitation of autophagosomes of the entire acinar cell volume (determined as a percentage as in A. Plotted as means ± SEM of 2 individuals per group. *P < vs pair-fed rats 24 hours after LPS; **P < vs alcohol-fed rats; ***P < .043 vs alcohol-fed rats 24 hours after LPS. (C) Representative immunofluorescence image of pair-fed control and alcohol-fed rats 24 hours after LPS rat tissues stained with LC3-Cy5 (red) using a confocal microscope with a 100× magnification. Arrowheads indicate nuclei; arrows indicate LC3-positive vesicle. (D) Representative immunoblot of LC3-I/LC3-II und Erk-2. The quantitation of the normalized ratio was plotted as means ± SEM of 4 animals per group. *P = vs pair-fed rats 24 hours after LPS; **P = vs pair-fed rats 24 hours after LPS; ***P = vs alcohol-fed rats 24 hours after LPS. (E) Representative EM image of pancreatic acinar cells exposed to alcohol for 14 weeks, 24 hours after LPS. Arrows indicate typical autolysosomes containing digested mitochondria and other intracellular components. (F) Quantitation of the volume of autolysosomes (expressed as percentage of the total acinar cell volume). Plotted as means ± SEM of 4 individuals per group. *P < vs pair-fed rats 24 hours after LPS; **P < vs alcohol-fed rats; ***P < vs alcohol-fed rats 24 hours after LPS; and ****P = vs alcohol-fed rats 24 hours after LPS.
Gastroenterology  , e5DOI: ( /j.gastro )
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7. Figure 6
Evaluation of lysosomal Lamp-2 and Gramp-92 expression in experimental pancreatitis. (A) Representative immunoblot of Lamp-2 und Erk-2. Normalized ratios were plotted as means ± SEM of 4 animals per group. *P = vs pair-fed rats 3 hours after LPS; **P = vs pair-fed rats 24 hours after LPS; ***P = vs alcohol-fed rats 24 hours after LPS. (B) Representative double-color immunofluorescence microphotographs of Lamp-2 and Gramp-92. Control and treated (alcohol plus LPS) samples were stained for Lamp-2 (green) and Gramp-92 (red) and counterstained with DAPI (blue). Yellow areas indicated colocalization of Lamp-2 and Gramp-92. Arrowheads indicate DAPI-stained nuclei; arrows indicate apical domains of acinar cells. (C) Quantitation of the fluorescence intensity of Lamp-2 (as percentage of DAPI intensity) expressed as means ± SEM of 2 representative individual animals per group. *P = .004 vs alcohol-fed rats 24 hours after LPS. (D) Quantitation of the fluorescence intensity of Gramp-92 normalized as presented in C. *P = vs alcohol-fed rats 24 hours after LPS. (E) Quantitation of the colocalization of Lamp-2 and Gramp-92. *P = vs alcohol-fed rats 24 hours after LPS.
Gastroenterology  , e5DOI: ( /j.gastro )
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8. Figure 7
Expression of Lamp-2 and LC3-I/-II after Lamp-2 silencing in AR42J cells. (A) Representative immunofluorescence microphotographs of Lamp-2 and LC3-I/-II in AR42J cells. Cells were stained for Lamp-2 (red) and LC3-I/-II (green) in controls (untreated and untreated plus control siRNA), after transfection with a Lamp-2-specific siRNA, alone or together with starvation. Nuclei were stained with DAPI (blue). Note the total loss of Lamp-2 positivity (red) and the appearance of additional cytoplasmic LC3-positive puncta (green) after Lamp-2 silencing. (B) Quantitation of Lamp-2 puncta per cell. Plotted as means ± SEM of >2 images per group with a minimum of 8 cells for each calculation. *P < vs Lamp-2 siRNA; **P < vs Lamp-2 siRNA plus starvation. (C) Quantitation of LC3 puncta per cell as in B. Plotted as means ± SEM of >2 images per group. *P < vs Lamp-2 siRNA; **P < vs Lamp-2 siRNA; ***P < .008 vs Lamp-2 siRNA plus starvation.
Gastroenterology  , e5DOI: ( /j.gastro )
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9. Figure 8
Evaluation of Lamp-2 and Lamp-1 expression in human pancreatitis specimens. Expression of Lamp-2 was determined in human pancreatitis and pancreatic donor tissue homogenates by immunoblot analysis and immunofluorescence. (A) Representative immunoblot of the Lamp-2 und Erk-2. Lanes 1–3: Pancreatic donor tissue; lanes 4–6: chronic alcoholic pancreatitis. (B) Representative immunofluorescence images for colocalization analysis were taken form human healthy controls and pancreatitis cryosections, stained for Lamp-2 (red), Lamp-1 (green), and DAPI (blue). Yellow areas indicate the colocalization of Lamp-2 and Lamp-1 and Lamp-2. Arrowheads indicate DAPI-stained nucleus, and arrows indicate the apical domain of acinar cells. (C and D) Quantitation of the immunofluorescence intensity of Lamp-2 (C) and Lamp-1 (D). Percentage intensity was plotted as means ± SEM of 2 individual patients per group. *P = (C) and P = .008 (D) vs pancreatitis. (E) Quantitation of the colocalization of Lamp-2 and Lamp-1 from healthy controls and pancreatitis patients expressed as means ± SEM of representative 2 individual patients per group. *P = vs pancreatitis tissue.
Gastroenterology  , e5DOI: ( /j.gastro )
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10. Supplementary Figure 1
Histopathology evaluation of pancreatic tissue in response to pair-fed and alcohol-fed rats 3 and 24 hours after LPS injection. Representative tissue sections stained with H&E. Alcohol-fed rats, killed 24 hours after LPS injection. Note the large area of vacuoles within this tissue.
Gastroenterology  , e5DOI: ( /j.gastro )
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11. Supplementary Figure 2
Electron microscopy evaluation of the involvement of mitochondria damage in pancreatic acinar cells after ethanol and endotoxemia. Images represent micropathologic alteration of pancreatic sections as described in the Material and Methods section. (A) Representative EM image of a pancreatic acinar cell. Arrow indicates damaged outer mitochondrial membrane and sign of partial mitochondrial lysis. (B) Higher magnification from the selected square area from the injured mitochondria as shown in A, indicating sign of calcification (arrowhead) and injured cristae (open arrow).
Gastroenterology  , e5DOI: ( /j.gastro )
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12. Supplementary Figure 3
Immunefluorescence evaluation of anti-Lamp-2 specific antibody in pancreatic acinar cells in pair-fed controls. Image represents Lamp-2 content of pancreatic sections as described in the Material and Methods section. Arrowhead indicates DAPI (blue)-stained acinar cell nuclear. Arrow indicates the apical domain of the acinar cells stained with Cy3 (green).
Gastroenterology  , e5DOI: ( /j.gastro )
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