1. Potential role of protease-activated receptor-2-stimulated activation of cytosolic phospholipase A2 in intestinal myofibroblast proliferation: Implications for stricture formation in Crohn's disease 
Ulrika Christerson, Åsa V. Keita, Johan D. Söderholm, Christina Gustafson-Svärd 
Journal of Crohn's and Colitis 
Volume 3, Issue 1, Pages (February 2009)
DOI: /j.crohns
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

2. Figure 1
Immunolabelling (green) of PAR-2 in ileal specimens from (a) a control patient with colonic cancer, and (b) microscopically uninflamed ileum of a patient with Crohn's ileitis. E=epithelium, MM=muscularis mucosae, SM=submucosa. Note the prominent PAR-2 expression of the expanded muscularis mucosae of the Crohn patient (b). Isotype-matched irrelevant negative control antibody showed no staining (c). Confocal images at a magnification of ×200. Scale bar=30 μm. Image a and b represent typical results obtained in five control patients and four Crohn patients, respectively. Note, however, that the increased epithelial PAR-2 expression suggested in panel b was not shown in all CD samples investigated.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

3. Figure 2
RT-PCR analysis of PAR-2 receptor mRNA expression in human intestinal myofibroblasts (CCD-18Co). PCR was performed using primers to amplify either human PAR-2 or 18SrRNA (used as house keeping gene). The PCR-products were identified as PAR-2 (899 bp) or 18SrRNA (531 bp) by comparison with a DNA ladder run in parallel (M). The identity of the amplified PAR-2 product was also verified by sequencing. (a) Demonstration of constitutive PAR-2 expression in pre-confluent cells and in confluent, nondividing, cells. RNA controls, containing untranscribed RNA (–), showed no genomic DNA contamination in the RNA preparations. The Caco-2 cell line was used as positive control (+) for PAR-2 expression. (b) Demonstration of increased PAR-2 mRNA expression in pre-confluent cells, and in confluent, non-dividing cells, incubated with TNF-α (25 ng/ml) for 24 or 48 h. To simplify the detection of any increased PAR-2 mRNA expression after TNF-α stimulation, the PCR-reaction was adjusted so that the expression in unstimulated cells was just below the detection limit in this set of experiments. Results are representative of three separate experiments.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

4. Figure 3
PAR-2 protein expression in human intestinal myofibroblasts (CCD-18Co). (a) PAR-2 immunolabelling (green) of cells incubated with TNF-α for 72 h, demonstrating an increased expression of PAR-2 compared to unstimulated control cells (b). Isotype-matched irrelevant negative control antibody showed no staining (c). For visualization of cells, actin filaments were stained with rhodamine–phalloidin (red). Confocal images, representative of results obtained in three separate experiments.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

5. Figure 4
Release of radiolabeled arachidonic acid (14C-AA) from human intestinal myofibroblasts (CCD-18Co), stimulated for 10 min with various concentrations of trypsin. Data from three to seven independent experiments. **p<0.01, ***p<0.001, versus cells stimulated with culture medium only (control). #p<0.05, ###p<0.01, versus cells stimulated with 1 or 10 nM trypsin.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

6. Figure 5
Effect of TNF-α on PAR-2-stimulated release of radiolabeled arachidonic acid (14C-AA) from human intestinal myofibroblasts (CCD-18Co). Cells were pre-treated with TNF-α for 72 h, before stimulated with PAR-2 activators for 10 min. Control cells were treated with culture medium only. (a) Effect of different TNF-α concentrations on the release of 14C-AA from cells stimulated with trypsin (100 nM). Data from five independent experiments. *p<0.05, ***p<0.001, versus control cells. (b) Effect of TNF-α (25 ng/ml) on the release of 14C-AA from cells stimulated with different trypsin concentrations. Data from three independent experiments. *p<0.05, ***p<0.001, versus control cells. (c) Effect of TNF-α (25 ng/ml) on the release of 14C-AA from cells stimulated with the PAR-2 activating peptide SLIGRL-NH2 (100 μM). Data from three independent experiments. ***p<0.001 versus control cells.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

7. Figure 6
Effect of PLA2 inhibitors on trypsin-stimulated release of radiolabeled arachidonic acid (14C-AA) from TNF-α-pre-treated human intestinal myofibroblasts (CCD-18Co). Cells were pretreated with TNF-α (25 ng/ml) for 72 h, and then incubated with the PLA2 inhibitor for 30 min, before being stimulated with trypsin (100 nM) for 10 min. Control cells were pre-treated with TNF-α, but otherwise treated with culture medium only. (a) Effect of the combined cPLA2 and iPLA2 inhibitor MAFP (10 μM). Data from five independent experiments. ***p<0.001 versus control cells. (b) Effect of the specific iPLA2 inhibitor BEL (10 μM). Data from four independent experiments. ***p<0.001 versus control cells.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

8. Figure 7
Stimulation of cPLA2 activity in human intestinal myofibroblasts (CCD-18Co). Cells were incubated with PMA (200 nM) for 1 h, and then with A23187 (4 μM) for further 10 min. Control cells were incubated with culture medium only. (a) Effect on the release of radiolabeled arachidonic and oleic acid. Data from eight (arachidonic acid) and four (oleic acid) independent experiments. ***p<0.001 versus control cells. (b) Effect of pre-treatment with the combined cPLA2 and iPLA2 inhibitor MAFP (10 μM) for 30 min. Data from four independent experiments. ***p<0.001 versus control cells. (c) Effect of pretreatment with TNF-α (25 ng/ml) for 72 h. Data from four independent experiments. ***p<0.001 versus control cells.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions

9. Figure 8
Effect of PLA2 inhibitors and trypsin on the growth of human intestinal myofibroblasts (CCD-18Co). Control cells were incubated with culture medium only. (a) Cells in five culture dishes were grown in the presence (10 μM) of a combined cPLA2 and iPLA2 inhibitor (AACOCF3), or a specific iPLA2 inhibitor (BEL). Control cells were incubated with culture medium only. After 3 days, cells were pulse-labeled with 3H-thymidin for 4 h. The number of cells per dish was determined in parallel cultures. Data from one of three independent experiments showing similar results. ***p<0.001 versus control cells. (b) Cells were serum-starved for 2 days and then incubated with trypsin (1 nM), with and without 5 μM AACOCF3, for 5 days. Data from four to nine independent experiments (all four treatment groups were included in four out of nine experiments, whereas five experiments included controls and trypsin-stimulated cells only). **p<0.01 versus control cells.
Journal of Crohn's and Colitis 2009 3, 15-24DOI: ( /j.crohns )
Copyright © 2009 European Crohn's and Colitis Organisation Terms and Conditions