1. Volume 143, Issue 5, Pages 1352-1360.e7 (November 2012)
NFATc3 Regulates Trypsinogen Activation, Neutrophil Recruitment, and Tissue Damage in Acute Pancreatitis in Mice 
Darbaz Awla, Anna V. Zetterqvist, Aree Abdulla, Cristina Camello, Lisa M. Berglund, Peter Spégel, Maria J. Pozo, Pedro J. Camello, Sara Regnér, Maria F. Gomez, Henrik Thorlacius 
Gastroenterology 
Volume 143, Issue 5, Pages e7 (November 2012)
DOI: /j.gastro
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2. Figure 1
Luciferase levels (relative luciferase units [RLU]/μg protein) in (A) pancreas, (B) aorta, (C) lung, and (D) spleen in age-matched adult NFAT-luc mice. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (gray bars) were infused with saline alone. Animals were pretreated with IP injections of the NFAT blocker A (0.15 mg/kg) or vehicle (saline) twice daily for 1 week before induction of pancreatitis. Samples were collected 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 6. #P < .05 versus control mice and *P < .05 versus taurocholate without A
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3. Figure 2
Quantitative measurements of (A) blood amylase (μKat/L), (B) pancreatic MPO (U/g tissue), (C) lung MPO (U/g tissue), (D) pancreatic CXCL2 (pg/mg tissue), and (E) pancreatic TAP (μg/g tissue) levels in age-matched adult NFAT-luc mice. Pancreatitis (black bars) was induced by infusion of sodium taurocholate into the pancreatic duct. Control mice (gray bars) were infused with saline alone. Animals were pretreated with IP injections of A (0.15 mg/kg) or vehicle (saline) twice daily for 1 week before induction of pancreatitis. An additional control group (white bars) received A (0.15 mg/kg) without bile duct cannulation. Samples were collected 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 6. #P < .05 versus control mice and *P < .05 versus taurocholate without A
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4. Figure 3
(A) Representative H&E-stained sections of the head of the pancreas from age-matched adult NFAT-luc mice. Black bars represent taurocholate-infused mice, and gray bars represent control mice infused with saline alone. Animals were pretreated with A (0.15 mg/kg) or vehicle (saline) as explained in Figure 2. Histologic quantification of (B) acinar cell necrosis, (C) edema, and (D) hemorrhage is shown from samples collected 24 hours after induction of pancreatitis. Scale bar = 100 μm. Data represent means ± SEM and n = 6. #P < .05 versus control mice and *P < .05 versus taurocholate without A
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5. Figure 4
Expression of NFATc1-c4 isoforms and of the housekeeping gene GAPDH was determined using RT-PCR in isolated pancreatic acini and lobules from young (Y, 18–22 days) and adult (A, 5–6 weeks) mice. Thymus (T) and spleen (S) were used as positive controls. The expected sizes of the products are as follows: NFATc1, 416 base pairs; NFATc2, 452 base pairs; NFATc3, 499 base pairs; NFATc4, 649 base pairs; and GAPDH, 293 base pairs. *300–base pair marker; **500–base pair marker. (F) Quantitative RT-PCR data showing NFATc1–c4 messenger RNA expression normalized to GAPDH in acini from adult NFATc3+/+ and NFATc3−/− mice. Data represent means ± SEM and n = 4.
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6. Figure 5
Quantitative measurements of (A) blood amylase (μKat/L), (B) pancreatic MPO (U/g), (C) lung MPO (U/g), (D) pancreatic CXCL2 (pg/mg), and (E) pancreatic TAP (μg/g) levels in adult NFATc3+/+, NFATc3+/−, and NFATc3−/− mice. Pancreatitis (black bars) was induced by infusion of sodium taurocholate into the pancreatic duct. Control mice (gray bars) received saline alone. Samples were obtained 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 8. #P < .05 versus control mice and *P < .05 versus NFATc3+/+ mice infused with taurocholate.
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7. Figure 6
(A) Representative H&E-stained sections of the head of the pancreas from NFATc3+/+, NFATc3+/−, and NFATc3−/− mice 24 hours after induction of pancreatitis with taurocholate and from control NFATc3+/+ and NFATc3+/− mice infused with saline alone. Histologic quantification of (B) acinar cell necrosis, (C) edema, and (D) hemorrhage. Scale bar = 100 μm. Data represent means ± SEM and n = 8. #P < .05 versus control NFATc3+/+ mice and *P < .05 versus taurocholate-infused NFATc3+/+ mice.
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8. Figure 7
In vitro measurements of trypsinogen activation in acinar cells from NFATc3−/− mice and NFATc3+/+ mice under control nonstimulated conditions (light gray bars) or after stimulation with CCK (100 nmol/L; black bars) or taurocholate (1%; dark gray bars) for 30 minutes. Cells were pretreated for 20 minutes and during the 30-minute stimulation period with either the NFAT blocker A (1 μmol/L) or vehicle (physiological solution). Trypsinogen activation was determined by measuring enzymatic activity of trypsin fluorometrically using Boc-Glu-Ala-Arg-MCA as the substrate as described in detail in Supplementary Materials and Methods. Trypsin levels (relative trypsin units [RTU]/pg) were calculated using a standard curve generated by assaying purified trypsin, normalized to protein concentration. Data represent means ± SEM and n = 5. #P < .05 versus control cells and *P < .05 versus CCK alone.
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9. Supplementary Figure 1
Luciferase levels (relative luciferase units per microgram protein) in (A) pancreas, (B) aorta, (C) lung, and (D) spleen in age-matched adult NFAT-luc mice. Pancreatitis (black bars) was induced by IP administration of l-arginine. Control mice (gray bars) received saline alone. Animals were pretreated with IP injections of the NFAT blocker A (0.15 mg/kg) or vehicle (saline) twice daily for 1 week before induction of pancreatitis. Samples were collected 72 hours after injection of l-arginine or saline. Data represent means ± SEM and n = 4. #P < .05 versus saline control; *P < .05 versus l-arginine without A
Gastroenterology  , e7DOI: ( /j.gastro )
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10. Supplementary Figure 2
Quantitative measurements of (A) blood amylase levels (μKat/L), (B) pancreatic MPO (U/g tissue), (C) lung MPO (U/g tissue), and (D) pancreatic edema in age-matched adult NFAT-luc mice. Pancreatitis (black bars) was induced by IP administration of l-arginine. Control mice (gray bars) received saline alone. Animals were pretreated with IP injections of A (0.15 mg/kg) or vehicle (saline) twice daily for 1 week before induction of pancreatitis. Samples were collected 72 hours after injection of l-arginine or saline. Data represent means ± SEM and n = 4. #P < .05 versus saline control; *P < .05 versus l-arginine without A
Gastroenterology  , e7DOI: ( /j.gastro )
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11. Supplementary Figure 3
(A) Representative H&E-stained sections of the head of the pancreas from age-matched adult NFAT-luc mice. Animals were treated IP with the NFAT blocker A (0.15 mg/kg) or vehicle (saline) before IP administration of l-arginine. Control mice received saline alone. (B) Histologic quantification of acinar cell necrosis in samples collected 72 hours after injection of l-arginine (black bars) or saline (gray bar). Scale bar = 100 μm. Data represent means ± SEM and n = 4. #P < .05 versus saline control; *P < .05 versus l-arginine without A
Gastroenterology  , e7DOI: ( /j.gastro )
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12. Supplementary Figure 4
Quantitative measurements of (A) blood amylase levels (μKat/L), (B) pancreatic MPO (U/g tissue), (C) lung MPO (U/g tissue), and (D) pancreatic edema in adult NFATc3+/+ and NFATc3−/− mice. Pancreatitis (black bars) was induced by IP administration of l-arginine. Control mice (gray bars) received saline alone. Samples were obtained 72 hours after injection of l-arginine or saline. Data represent means ± SEM and n = 5. #P < .05 versus saline control; *P < .05 versus NFATc3+/+.
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13. Supplementary Figure 5
Representative H&E-stained sections of the head of the pancreas from adult NFATc3+/+ and NFATc3−/− mice. Pancreatitis was induced by IP administration of l-arginine. Control mice received saline alone. (B) Histologic quantification of acinar cell necrosis. Samples were collected 72 hours after injection of l-arginine (black bars) or saline (gray bar). Scale bars = 100 μm. Data represent means ± SEM and n = 5. #P < .05 versus saline control; *P < .05 versus NFATc3+/+.
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14. Supplementary Figure 6
(A) Representative H&E-stained pancreas sections from NFATc3+/+ and NFATc3−/− mice. (B) Blood glucose levels and (C) body weight of adult NFATc3+/+ (black bars) and NFATc3−/− (white bars) mice. Scale bar = 100 μm and n = 19.
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15. Supplementary Figure 7
(A–F) Expression of inflammatory genes in acinar cells from adult NFATc3+/+ (+/+) or NFATc3−/− (−/−) mice under control nonstimulated conditions (gray bars) or after stimulation with CCK (100 nmol/L, black bars) for 60 minutes. Cells were pretreated for 20 minutes and during the 60-minute stimulation period with the NFAT blocker A (0.1 μmol/L), the calcineurin inhibitor CsA (10 μmol/L), or vehicle (PS). Expression levels of COX-2, IL-6, IL-1β, MCP-1, CXCL2, and MMP9 messenger RNA were measured by quantitative RT-PCR using GAPDH as endogenous control. Data represent means ± SEM and n = 10 and 14 for NFATc3+/+ and NFATc3−/−, respectively. For NFATc3+/+: #P < .05 vs control cells and *P < .05 or **P < .01 vs CCK alone; for NFATc3−/−: **P < .01 or ***P < .001 vs control cells.
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16. Supplementary Figure 8
(A) Confocal immunofluorescence images showing predominantly cytosolic distribution of endogenous NFATc3 (green) under basal nonstimulated conditions (control, left panels) and increased NFATc3 nuclear accumulation in response to 10 nmol/L CCK stimulation for 7 minutes (right panels). PI-stained nuclei are shown in red and colocalization of the signals in the merged images in white. Scale bar = 10 μm. Summarized confocal data showing NFATc3 nuclear accumulation after stimulation with (B) 10 nmol/L CCK for 7, 20, and 50 minutes (331, 106, and 101 cells were examined from 8, 4, and 4 mice, respectively, for each time point); (C) 10 pmol/L CCK for 7, 15, or 30 minutes (121, 687, and 103 cells were examined from 4, 12, and 4 mice, respectively, for each time point); and (D) 10 μmol/L acetylcholine for 7, 20, and 50 minutes (426, 185, and 142 cells were examined from 12, 4, and 4 mice, respectively, for each time point). (E) Agonist-induced NFATc3 nuclear accumulation was prevented by preincubation for 30 minutes with CsA (10 μmol/L). Incubation for 10 minutes with ionomycin (10 μmol/L) resulted in increased NFATc3 nuclear accumulation, while CsA alone had no effect. At least 120 cells were examined, and 4–12 mice were used in each group. Data are expressed as fold of controls in all graphs. #P < .05 versus control and *P < .05 for “agonist + CsA”-treated cells versus corresponding agonist alone.
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17. Supplementary Figure 9
Summarized data from confocal immunofluorescence experiments showing NFATc1 nuclear accumulation after stimulation with (A) 10 nmol/L CCK for 7, 20, and 50 minutes (231, 207, and 169 cells were examined from 6, 4, and 4 mice, respectively, for each time point); (B) 10 pmol/L CCK for 7, 15, or 30 minutes (481, 134, and 187 cells were examined from 6, 4, and 2 mice, respectively, for each time point); and (C) 10 μmol/L acetylcholine for 7, 20, and 50 minutes (222, 182, and 169 cells were examined from 6, 4, and 4 mice, respectively, for each time point). (D) Agonist-induced NFATc1 nuclear accumulation was prevented by preincubation for 30 minutes with CsA (10 μmol/L). Incubation with ionomycin (10 μmol/L, 10 minutes) increased NFATc1 nuclear accumulation, while CsA alone yielded levels below those of control. At least 110 cells were examined, and 4 mice were used in each group. Data are expressed as fold of controls in all graphs. #P < .05 versus control and *P < .05 for “agonist + CsA”-treated cells versus corresponding agonist alone.
Gastroenterology  , e7DOI: ( /j.gastro )
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