1. Volume 144, Issue 1, Pages 192-201 (January 2013)
Deletion of IκBα Activates RelA to Reduce Acute Pancreatitis in Mice Through Up- regulation of Spi2A 
Patrick Neuhöfer, Song Liang, Henrik Einwächter, Christiane Schwerdtfeger, Thomas Wartmann, Matthias Treiber, Hong Zhang, Hans– Ulrich Schulz, Karen Dlubatz, Marina Lesina, Kalliope N. Diakopoulos, Sonja Wörmann, Walter Halangk, Heiko Witt, Roland M. Schmid, Hana Algül 
Gastroenterology 
Volume 144, Issue 1, Pages (January 2013)
DOI: /j.gastro
Copyright © 2013 AGA Institute Terms and Conditions

2. Figure 1
Pancreas-specific deletion of IκBα results in nuclear translocation of RelA and a subsequent NF-κB activation. (A) Immunohistochemical detection of phospho-IκBα during pancreatitis at the indicated time points. Note the increase of phospho-IκBα–positive cells (black arrows) after 1 hour. Inset: positive staining of the spleen. (B) Nuclear localization of RelA in the pancreas of IκBα-deficient mice. Nuclear (10 μg) and cytosolic (20 μg) extracts from isolated acini were analyzed using the indicated antibodies. (C) NF-κB/Rel binding activity in pancreata of unstimulated IkbaF/F and IkbaΔpanc mice. Competition experiment. Binding activity in the presence of 20-fold unlabeled oligonucleotide. (D) Pancreatic cryosections of IkbaΔpanc were analyzed for nuclear localization of RelA. Nuclei were stained with Sytox green (Life Technologies GmbH, Darmstadt, Germany). Localization of RelA was visualized in red. Nuclear localization of RelA appears in orange. The dashed line marks an islet. (E) Gene expression analysis of 8-week-old pancreata from wt, RelaΔpanc, and IkbaΔpanc mice. Analysis of an NF-κB–specific gene set (V$NFKAPPAB_01) using GSEA software (Broad institute, Cambridge, MA) showed a significant up-regulation in IkbaΔpanc samples and a down-regulation in RelaΔpanc samples (each tested against wild type). (F) Real-time polymerase chain reaction of different cytokines and chemokines in pancreatic tissue of IkbaF/F and IkbaΔpanc mice. Results represent fold-change against wt (n = 3). Statistical significance is shown: *P < .05, **P < .005 vs control mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

3. Figure 2
Acute pancreatitis is attenuated in mice with pancreas-specific deletion of IκBα. (A) Representative electrophoretic mobility shift assay showing NF-κB/Rel binding activity in cerulein pancreatitis in IkbaF/F and IkbaΔpanc mice. Graphic representation of densitometry analysis (n = 4). (B) Western blot analysis of NF-κB proteins of IkbaF/F and IkbaΔpanc mice at different time points. Extracellular-signal-related kinase (Erk) 1/2 served as loading control (n = 3). (C) Mice were treated with a total of 8 hourly intraperitoneal injections of cerulein and were killed at indicated time points. Histologic sections of the pancreas of IkbaF/F and IkbaΔpanc mice were analyzed. Note the increased edema (black stars) in IkbaF/F mice. (D and E) Serum amylase and lipase levels were measured in IkbaF/F and IkbaΔpanc mice (n > 4). (F) Pancreatic weight related to body weight (relative pancreatic weight) was evaluated (n > 4). *P < .05 and ***P <.001 vs IkbaF/F control mice was considered significant.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

4. Figure 3
Trypsin activity is decreased in IkbaΔpanc mice during AP. Trypsin activity was measured at the indicated time points in IkbaF/F and IkbaΔpanc mice (n = 4). Results are means ± standard deviation. **P < .005 was considered significant.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

5. Figure 4
Pancreas-specific ablation of IκBα attenuates pancreatitis-associated lung complication. (A) Expression of IκBα in lung tissue of IkbaF/F and IkbaΔpanc mice was assessed. β-actin served as a loading control (representative blot, n = 3). (B) Lung tissue was removed and stained at the indicated time points. A higher magnification (200×) of representative sections reveals markedly decreased hemorrhage and alveolar collapse in IkbaΔpanc mice. (C) Lung tissue was used to determine myeloperoxidase enzyme activity (n = 5). (D) Interleukin (IL)-6 serum levels in IkbaF/F and IkbaΔpanc mice were determined by enzyme-linked immunosorbent assay (n = 4). Asterisks indicate statistical significance: *P < .05 vs IkbaF/F control mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

6. Figure 5
Protective effects in IkbaΔpanc mice during AP depend on nuclear RelA. (A) Supershift assay identifies the RelA/p65 subunit as part of the NF-κB complex in IkbaΔpanc mice during AP (1 h). (B) Acini from mice with indicated genotype were isolated. Cytosolic protein lysates were analyzed using antibodies directed against IκBα and RelA. In contrast to IkbaF/F or IkbaΔpanc mice, acini isolated from Ikba x RelaΔpanc double-knockout mice displayed a truncated form of RelA, termed ΔRelA. (C) Histologic sections of the pancreas of IkbaF/F, IkbaΔpanc, and Ikba x RelaΔpanc mice were analyzed after 8 and 24 hours of cerulein administration. Ikba x RelaΔpanc mice showed increased edema (black stars) and necrosis (dashed line). (D) Sections were scored from 0 (normal) to 3 (severe) for inflammation (n = 4). (E) Sections were analyzed to evaluate necrosis in relation to total pancreatic area (n = 3). (F) Serum amylase activity was measured. Note the significantly lower release of amylase into the serum of IkbaΔpanc mice and the increased release in the case of Ikba x RelaΔpanc mice compared with IkbaF/F mice. All values are means ± standard deviation for independent animals (n = 5). Statistically significant: *P < .05 and **P < .005 vs IkbaF/F control mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

7. Figure 6
Spi2A expression was up-regulated in IκBα-deficient mice. (A) Expression analysis of 8-week-old pancreata from IkbaF/F, RelaΔpanc, and IkbaΔpanc mice. Heat map of significantly regulated transcripts between IκBα-deficient and other pancreata. (B) Pancreata from IkbaF/F, IkbaΔpanc, and RelaΔpanc mice were isolated and homogenized to detect Spi2A, IκBα, RelA, and truncation of RelA (ΔRelA) by Western blot. β-actin served as loading control (n = 4). (C) Total pancreatic lysates and acinar cells from IkbaF/F, IkbaΔpanc mice were subjected to Spi2A expression analysis by Western blot. (D) In vivo and in vitro stimulated acinar cells were analyzed for Spi2A expression by Western blot. NS, nonspecific band. (E) Chromatin immunoprecipitation experiments were performed with pancreatic tissue of the indicated genotypes using an antibody against RelA. Precipitated DNA was analyzed by polymerase chain reaction using primers surrounding the positions of a κB site in the Spi2a promoter 7.2 kB from the 5' end of exon 1. Polymerase chain reaction also was performed with 2.5% of input chromatin to ensure equal loading.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

8. Figure 7
Protective effects of Spi2A in cerulein-induced pancreatitis. (A) Model of lentiviral transduction. C57BL/6 mice were injected (intraperitoneally) with lentiviral vectors on days 1 and 4. On day 14 the mice were challenged with 8 hourly injections of cerulein. (B) Immunoblot analysis. Pancreas from mice, either injected with LV-Spi2A or LV-empty, was isolated to detect Spi2A. Note the increased expression in mice injected with the LV-Spi2A construct. Extracellular-signal-related kinase (ERK) 1/2 served as loading control (representative blot, n = 3). (C) Histologic sections of the pancreas of LV-Spi2A and LV-empty mice were analyzed at the indicated time points. Note the increased necrosis (dashed line) and edema (black stars) in LV-empty mice. (D and E) Sections were analyzed to evaluate necrosis and edema in relation to total pancreatic area. (F) Trypsin activity was measured after 1 and 8 hourly injections in LV-Spi2A and LV-empty mice during AP. Note the significantly low activity of trypsin in LV-Spi2A compared with LV-empty mice after 1 and 8 hours. Results are means ± standard error (n > 3 for each group). *P < .05; **P < .005, and ***P < .001 was considered significant.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions