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 350-360.e5 (July 2009) DOI: 10.1053/j.gastro.2009.04.003 Copyright © 2009 AGA Institute Terms and Conditions

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 = .0188 vs pair-fed rats 24 hours after LPS (necrosis); **P = .0005 vs alcohol-fed rats 24 hours after LPS (apoptosis); ***P = .0063 vs alcohol-fed rats 24 hours after LPS (necrosis); ****P = .0312 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 = .0489 vs alcohol-fed rats 3 hours after LPS injection; #P = .0824 vs alcohol-fed rats 3 hours after LPS injection. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 < .0001 vs pair-fed control rats; **P < .0001 vs pair-fed control; and ***P = .0043 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 = .0351 vs alcohol plus LPS; #P = .0523 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 = .0185 vs alcohol-fed rats 24 hours after LPS injection. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 < .0001 vs pair-fed animals 24 hours after LPS; **P = .0309 vs alcohol-fed animals; ***P = .0095 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 = .0222 vs alcohol-fed rats; **P = .0412 vs alcohol-fed rats 24 after LPS. (D) Representative immunoblot of pro- and active caspase-9. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 = .0013 vs pair-fed rats 24 hours after LPS; **P = .0249 vs alcohol-fed rats; ***P < .0001 vs alcohol-fed rats 24 hours after LPS; ****P = .0423 vs alcohol-fed rats 24 hours after LPS. (B) Quantitation of trypsin activity was plotted as means ± SEM of 5 animals per group. *P < .0001 vs pair-fed rats 24 hours after LPS; **P = .0347 vs alcohol-fed rats; ***P < .0001 vs alcohol-fed rats 24 hours after LPS; ****P = .0406 vs alcohol-fed rats 24 hours after LPS. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 < .0008 vs pair-fed rats 24 hours after LPS; **P < .0043 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 = .0034 vs pair-fed rats 24 hours after LPS; **P = .0008 vs pair-fed rats 24 hours after LPS; ***P = .0169 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 < .0001 vs pair-fed rats 24 hours after LPS; **P < .0001 vs alcohol-fed rats; ***P < .0001 vs alcohol-fed rats 24 hours after LPS; and ****P = .0044 vs alcohol-fed rats 24 hours after LPS. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 = .0054 vs pair-fed rats 3 hours after LPS; **P = .0015 vs pair-fed rats 24 hours after LPS; ***P = .0222 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 = .0022 vs alcohol-fed rats 24 hours after LPS. (E) Quantitation of the colocalization of Lamp-2 and Gramp-92. *P = .0042 vs alcohol-fed rats 24 hours after LPS. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 < .0027 vs Lamp-2 siRNA; **P < .0217 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 < .0147 vs Lamp-2 siRNA; **P < .0393 vs Lamp-2 siRNA; ***P < .008 vs Lamp-2 siRNA plus starvation. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 = .0141 (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 = .0094 vs pancreatitis tissue. Gastroenterology 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions

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 2009 137, 350-360.e5DOI: (10.1053/j.gastro.2009.04.003) Copyright © 2009 AGA Institute Terms and Conditions