1. Volume 127, Issue 1, Pages 275-286 (July 2004)
Overexpression of heat shock protein Hsp27 protects against cerulein-induced pancreatitis 
Constanze Kubisch, Matthew J. Dimagno, Anne Barbara Tietz, Michael J. Welsh, Stephen A. Ernst, Barbara Brandt-Nedelev, Joachim Diebold, Andreas C.C. Wagner, Burkhard Göke, John A. Williams, Claus Schäfer 
Gastroenterology 
Volume 127, Issue 1, Pages (July 2004)
DOI: /j.gastro

2. Figure 1
Protein expression of Hsp27 in transgenic mice. (A) Immunoblot of protein lysates (30 μg protein/lane) using anti-huHsp27 antibody shows tissue distribution of huHsp27 (upper panel). Purified recombinant Hsp27 and Hsp25 protein (3 and 30 ng, respectively) was used as positive control. Stripping and reprobing of the membrane with anti-Hsp25 antibody showed tissue expression of endogenous Hsp25 protein (lower panel). Representative blots of at least 3 individual experiments are shown. (B) Localization of Hsp27 in the pancreas from transgenic mice by immunofluorescence. Fluorescence huHsp27 staining was strongly visualized in acini. Note that some acinar patches showed less staining (left panel). Immunofluorescence analysis of pancreas from nontransgenic animals showed no specific staining (right panel). Images are representative of those obtained from 3 independent experiments. (Original magnification 275×.)
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Screening of transgenic mice by PCR and Western blotting. After preparation of genomic DNA from animals, the incorporation of the transgene was tested after amplification with specific primers by PCR. (A) Representative amplified PCR products for all 3 transgenic mouse lines are shown (lanes 1–6). There was no gene product amplified from nontransgenic animals (lanes 7 and 8). Distilled water served as a negative control (lane 10) and purified DNA as a positive control (lane 9). The arrow indicates the specific gene product at 280 base pairs. (B, upper panel) Immunoblotting with anti-Hsp27 antibody. All transgenic animals express similar amounts of Hsp27 in the pancreas (lanes 3–8) (pancreas lysates containing 20 μg total protein/lane). Note that no expression of Hsp27 was observed from nontransgenic animals (lanes 1 and 2). (B, lower panel) Testing the same protein lysates as used in the upper panel, all samples showed nearly the same protein expression of endogenous Hsp25. In contrast to the upper panel, lanes 1 and 2 from nontransgenic animals showed expression of Hsp25, indicating the specificity of the antibodies used. The Western blots shown here are representative blots from at least 6 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Phosphorylation analysis of transgenic Hsp27. (A) Western blotting with a phospho-specific antibody to evaluate the phosphorylation of Ser78 of Hsp27 (upper panel). P, pancreas removed quickly without treatment of the animals; Cont., lysates from animals that received 12 hourly intraperitoneal injections of saline; Cer, 12 hourly intraperitoneal injections of 50 μg/kg cerulein. Lines 1–3 represent lysates from huHsp27 mice, lines 4–6 from huHsp27-3D, and lines 7–9 from huHsp27-3A. Western blotting with an anti-Hsp27 antibody that recognizes the total amount of huHsp27 showed similar amounts of protein in all samples (lower panel). (B) Phosphorylation analysis by isoelectric focusing and Western blotting. Representative immunoblots of at least 3 independent experiments are shown in A and B.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Effects of overexpression of Hsp27 and its mutants on cerulein-induced pancreatitis. (A) Serum amylase levels, (B) serum lipase levels, (C) pancreatic water content, and (D) trypsin activity were measured as described in the text after 12 hourly injections of 50 μg/kg cerulein (black bars). Control animals received saline (white bars). Results are expressed as mean ± SEM (number of animals per group is indicated). ∗P < 0.05, ∗∗P < 0.01 was considered significant.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Morphologic changes in cerulein-induced pancreatitis. Representative H&E-stained sections of pancreas were examined by light microscopy in (A) normal control nontransgenic animals, (B) an huHsp27 control animal, (C) a nontransgenic animal administered 12 intraperitoneal cerulein injections, (D) an huHsp27-3A animal administered 12 intraperitoneal cerulein injections, (E) an huHsp27 animal administered 12 intraperitoneal cerulein injections, and (F) an huHsp27-3D animal administered 12 intraperitoneal cerulein injections. The histologic scoring of these representative sections is as follows: edema 2.0 (C), 1.5 (D), 1.0 (E), 1.0 (F); inflammation 2.0 (C), 1.5 (D), 1.0 (E), 0.5 (F); vacuolization 2.0 (C), 2.0 (D), 1.0 (E), 0.5 (F); necrosis 2.0 (C), 2.0 (D), 0.5 (E), 0.0 (F).
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Effects of overexpression of Hsp27 and its mutants on pancreatic inflammatory changes following pancreatitis. Histologic sections of pancreata harvested 1 hour after 12 hourly injections of saline or cerulein (Cer) from nontransgenic mice (open bars), huHsp27-expressing transgenic mice (hatched bars), huHsp27-3D-expressing transgenic mice (lined bars), and huHsp27-3A-expressing transgenic mice (crossed bars) were scored from 0 (normal) to 3 (severe) for edema, inflammation, vacuolization, and necrosis as described in Materials and Methods. Results are expressed as mean ± SEM; ∗P < 0.05 versus saline-injected controls, †P < 0.05 versus nontransgenic mice. This figure shows one experiment testing 4–5 mice per group.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Effects of overexpression of Hsp27 and its mutants on filamentous actin visualized with phalloidin in pancreatic acinar cells. Animals were administered 12 injections of either saline or cerulein, and pancreata were subsequently processed as described in the text. In control pancreatic acini from (A) nontransgenic and (B) transgenic huHsp27 animals, actin was present as sharply demarcated bands in the terminal web region just beneath the acinar lumen (large arrows) and subjacent to the lateral and basal plasma membranes. Actin was also present in the duct and centroacinar cells. Injection of cerulein induced a severe disruption of the actin-staining pattern in (C) nontransgenic and (D) huHsp27-3A animals. Subluminal actin was no longer apparent. Acinar cells with poorly organized or fragmented actin were often rounded up, and these cells generally expressed actin staining continuously along the cell periphery (arrowheads). In addition, diffuse cytoplasmic fluorescence was also present in variable degrees. In contrast, actin staining was only moderately changed in animals overexpressing (E) huHsp27 or (F) huHsp27-3D. Acini retained their polarized state, and actin organization was only moderately perturbed with some increase in cytoplasmic staining that was variable in amount. Images are representative of at least 4 individual animals in each group.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
MPO activity was measured in the lung after 12 hourly cerulein injections in nontransgenic mice (open bar), mice overexpressing huHsp27 (hatched bar), huHsp27-3D (lined bar), or huHsp27-3A (crossed bar). Control mice received saline injections (pooled data from nontransgenic and transgenic mice). Data are expressed as MPO activity (mU/mg tissue weight). ∗P < 0.05 versus control; †P < 0.05 versus nontransgenic mice. This figure shows data for 4–5 mice per group.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Western blot analysis of Hsp70. Induction of Hsp70 was detectable during development of pancreatitis (A, upper panel, lanes 2, 4, 6, and 8). Pancreatic lysate from a rat after exposure to hyperthermia served as a positive control (lane 9). No significant difference between transgenic and nontransgenic animals was observed. Washing of the membrane and incubating with an anti-Hsp25 antibody showed a similar expression of endogenous Hsp25 in all samples (A, lower panel). (B) Representative immunoblots of at least 3 independent experiments are shown. Expression of huHsp27 in the same samples after Western blotting with the specific huHsp27 antibody. Recombinant Hsp27 protein was used as positive control for the antibody.
Gastroenterology  , DOI: ( /j.gastro )