Leukotrienes induce cell-survival signaling in intestinal epithelial cells  John F. Öhd, Katarina Wikström, Anita Sjölander  Gastroenterology  Volume 119, Issue 4, Pages 1007-1018 (October 2000) DOI: 10.1053/gast.2000.18141 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 LTD4-induced accumulation of COX-2 in membrane fractions of Int 407 cells. (A and B) Representative Western blots of (A) COX-2 and (B) COX-1 in membrane fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. (C) Densitometric analysis of COX-2 levels in cells exposed to LTD4 for different periods in the absence (●) or presence (○) of the LTD4-receptor antagonist ZM198,615. (D) Concentration-dependent effects of LTD4 on COX-2 levels in cells exposed to different concentrations of LTD4 for 60 minutes in the absence (●) or presence (○) of ZM198,615. The densitometric values represent means ± SEM of 4 separate experiments. *P < 0.05; **P < 0.01. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 LTD4-induced accumulation of β-catenin in membrane and cytosolic fractions of Int 407 cells. (A) Representative Western blot of β-catenin in membrane (top panels) and cytosolic (lower panels) fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. As loading controls, we used the presence of transferrin receptors in the membrane fractions and the presence of actin in the cytosolic fractions. (B) Densitometric analysis of β-catenin levels in membrane fractions from cells exposed to LTD4 for different periods in the absence (●) or presence (○) of the LTD4-receptor antagonist ZM198,615. (C) Concentration-dependent effects of LTD4 on β-catenin levels in membrane fractions from cells exposed to different concentrations of LTD4 for 60 minutes in the absence (●) or presence (○) of ZM198,615. The densitometric values represent means ± SEM of 4 separate experiments. *P < 0.05; **P < 0.01. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 LTD4-induced accumulation of Bcl-2 in membrane fractions of Int 407 cells. (A) Representative Western blot of Bcl-2 in membrane fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. As loading control we used the presence of transferrin receptors in the membrane fractions. (B and C) Densitometric analysis of Bcl-2 protein levels in cells treated in the same way as described in Figures 1 and 2. The densitometric values represent means ± SEM of 4 separate experiments. *P < 0.05; **P < 0.01. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 LTD4-induced accumulation of COX-2, β-catenin, and Bcl-2 in membrane fractions of IEC-6 cells. The Western blots show the presence of COX-2, β-catenin, and Bcl-2 in membrane fractions of cells stimulated with 40 nmol/L LTD4 for the indicated periods. The Western blots were analyzed with specific antibodies against the respective proteins; the COX-2 antibody was obtained from Santa Cruz Biotechnology. The blots are representative of at least 3 separate experiments. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 The effects of 40 nmol/L LTB4 (●) and LTC4 (○) on accumulation of (A) COX-2, (B) β-catenin, and (C) Bcl-2 in membrane fractions of Int 407 cells. The data were obtained by densitometric analysis of the results and represent means ± SEM of 4 separate experiments. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of LTD4 and NS-398 on membrane accumulation of COX-2, β-catenin, and Bcl-2 and production of PGE2 in intestinal cells. The cells were preincubated for 30 minutes in the absence or presence of 100 μmol/L NS-398 before stimulation with 40 nmol/L LTD4 for 1 hour; untreated cells were used as controls. (A) Representative Western blots probed with specific antibodies against COX-2 (Santa Cruz), β-catenin, and Bcl-2. The blots shown are representative of at least 3 separate experiments. (B) PGE2 formation in Int 407 cells treated as described in A. (C) PGE2 formation in IEC-6 cells treated as described in A. The data in B and C are expressed as percent of control values and represent means ± SEM of 3 separate experiments. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Int 407 and (B) IEC-6 cell morphology visualized by phase-contrast microscopy. The micrographs show control cells and cells treated with 100 μmol/L NS-398 or 100 μmol/L NS-398 and 40 nmol/L LTD4. Each micrograph is representative of 4 separate experiments (original magnification 40×). Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 (A) Fluorescence micrographs of Int 407 cells stained with Hoechst 33342 (original magnification 60×). Control cells, cells treated with 100 μmol/L NS-398, and cells exposed to 100 μmol/L NS-398 and 40 nmol/L LTD4 are shown. (B) Percentage apoptotic nuclei (calculated as Hoechst-positive cells per total cells) in untreated cells (control) and cells exposed to NS-398 alone or with 40 nmol/L LTD4. (C) Results of the trypan blue viability assay in control cells and cells treated with LTD4, NS-398, or NS-398 and 40 nmol/L LTD4. The data are representative of 4 separate experiments and are means ± SEM. *P < 0.05; **P < 0.01. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 (A) Caspase-3 activity illustrated as percentage of baseline activity in lysates of Int 407 cells exposed to LTD4 and NS-398 for the indicated periods. The fluorescence intensity data represent means ± SEM of 4 separate experiments; *P < 0.05; **P < 0.01. (B) DNA fragmentation over 24 hours in Int 407 cells. Results are shown for untreated cells (lane 1) and for cells treated with 40 nmol/L LTD4 (lane 2), 100 μmol/L NS-398 (lane 3), and both NS-398 and LTD4 (lane 4). The pattern is representative of 4 separate experiments. Gastroenterology 2000 119, 1007-1018DOI: (10.1053/gast.2000.18141) Copyright © 2000 American Gastroenterological Association Terms and Conditions