1. Volume 119, Issue 3, Pages 816-828 (September 2000)
Tumor necrosis factor α triggers antiapoptotic mechanisms in rat pancreatic cells through pancreatitis-associated protein I activation 
David Malka, Sophie Vasseur, Hans Bödeker, Emilia M. Ortiz, Nelson J. Dusetti, Patrick Verrando, Jean–Charles Dagorn, Juan L. Iovanna 
Gastroenterology 
Volume 119, Issue 3, Pages (September 2000)
DOI: /gast
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A) TNF-α induces pancreatic acinar apoptosis in a time- and dose-dependent manner. AR4-2J cells were treated with TNF-α for varying incubation times (24, 48, and 72 hours) and at varying doses (1, 10, 50, 100, and 200 ng/mL). Cell viability was measured using a spectrophotometric MTS assay (see Materials and Methods). Cell viability was expressed as the percentage of optical density values for the corresponding samples to values for cells untreated with TNF-α. Values represent the mean of 3 experiments performed in quadruplicate. Maximal SD corresponds to 16% of the mean. (B) DNA release from AR4-2J cells treated with TNF-α. AR4-2J cells were prelabeled with BrdU (10 μg/mL) for 16 hours and plated in 96-well microtiter plates. Twenty-four hours later, cells were treated with TNF-α (100 ng/mL) (left panel) or TNF-α (100 ng/mL) in combination with actinomycin D (10 μg/mL) (right panel) for different incubation times, and the BrdU-labeled DNA fragments in supernatant and cell lysate were assayed using an ELISA test (see Materials and Methods). DNA release was expressed as the percentage of optical density values for the corresponding samples to values for cells untreated with TNF-α. Values represent the mean of 2 experiments performed in triplicate. Maximal SD corresponds to 15% of the mean. (C) Actinomycin D treatment enhanced the apoptotic effect of TNF-α on AR4-2J cells. AR4-2J cells were pretested with actinomycin D (10 μg/mL), then with TNF-α for varying incubation times (3, 6, 9, 12, 18, and 24 hours) and at varying doses (1, 5, 10, 20, and 100 ng/mL). Cell viability was measured; values are expressed as described earlier. Values represent the mean of 3 experiments performed in quadruplicate. Maximal SD corresponds to 19% of the mean. All values are within 2 SD of the mean.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

3. Fig. 2
TNF-α induces p65 nuclear translocation in AR4-2J cells. Cells were left (A) untreated or (C) transduced with the AdvIκBα recombinant adenovirus and (B and C) incubated with 100 ng/mL TNF-α for 1 hour. Indirect immunofluorescence staining using rabbit anti-p65 polyclonal antibodies (1:100), biotinylated goat anti-rabbit antibodies (1:350), and FITC-streptavidin conjugate (10 μg/mL) was performed, and visualized under a fluorescence microscope.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Pharmacologic inhibition of TNF-α–induced NF-κB translocation increases TNF-α–induced pancreatic acinar apoptosis. AR4-2J cells were pretested with varying concentrations of (A) PDTC, (B) ALLN, (C) SN50, or (D) sodium salicylate, and then treated with TNF-α (100 ng/mL) for 24, 48, or 72 hours. Cell viability was measured; values are expressed as described in Figure 1. Values represent the mean of 3 experiments performed in quadruplicate. Maximal SD corresponds to 18% of the mean. All values are within 2 SD of the mean.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Molecular inhibition of TNF-α–induced NF-κB translocation increases TNF-α–induced pancreatic acinar apoptosis. AR4-2J cells were transduced with 25, 50, 75, or 100 MOI of AdvIκBα (■) or AdvGFP (□, control) recombinant adenovirus and 72 hours later incubated with TNF-α (100 ng/mL) for 24 hours. Cell viability was measured as described in Figure 1. Cell viability was expressed as the percentage of optical density values for the corresponding samples to values for cells untreated with TNF-α and not infected with an adenovirus (mean of 2 experiments performed in quadruplicate).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

6. Fig. 5
(A) p38, ERK1/2 (p44/p42), and JNK (p54/p46) protein kinase activation in AR4-2J by TNF-α treatment. AR4-2J cells were treated with 100 ng/mL TNF-α (see Materials and Methods), and protein extracts were prepared 15 and 30 minutes after cytokine addition. (B) Low concentrations of TNF-α activate MAP kinases in AR4-2J cells. AR4-2J cells were treated with 1, 10, 50, or 100 ng/mL TNF-α for 30 minutes for p38 and ERK1/2 (p42/p44), or 15 minutes for JNK (p46/p54). Total cell protein (80 μg) was separated on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Western blot was revealed with the ECL system (Amersham Pharmacia Biotech, Les Ulis, France). Activity of stress-sensitive protein kinases was measured by Western blotting using specific antibodies against their active (phosphorylated) forms. The antibodies were polyclonal anti–phospho-p38 antibody, monoclonal anti–phospho-p44/p42 E10 antibody, and monoclonal anti–phospho-JNK G9 antibody.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Pharmacologic blocking of the MAP kinase activities increases TNF-α–induced pancreatic acinar apoptosis. AR4-2J cells were pretested with 2 concentrations (5 μmol/L [□] and 20 μmol/L [■]) of the MAP kinase inhibitors PD98059, SB203580, SB202190, or SB (control) and then treated with TNF-α (100 ng/mL) for 24 hours. Values are expressed as the percentage of the optical density values for the corresponding samples to values for cells untreated with TNF-α (mean of 3 experiments performed in quadruplicate).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

8. Fig. 7
PAP I expression increases resistance to TNF-α–induced apoptosis in AR4-2J cells. AR4-2J cells were transduced with 10 MOI of Ad.CMV/PAP (□) or Ad.CMV/CAT (■; control) recombinant adenovirus and 72 hours later incubated with TNF-α (100 ng/mL) in the presence of actinomycin D (10 μg/mL) and/or Z-VAD-FMK (20 μmol/L) for 12 hours. Cell viability was measured as described in Figure 1 and expressed as the percentage of the optical density values for the corresponding samples to values for cells untreated with TNF-α (mean of 3 experiments performed in quadruplicate).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

9. Fig. 8
PAP I expression is induced by low concentrations of TNF-α in AR4-2J cells. (A) AR4-2J cells were treated with 1, 10, 50, or 100 ng/mL TNF-α, and PAP I mRNA expression was measured by a semiquantitative RT-PCR approach. PAP I mRNA expression in response to TNF-α is inhibited by PD98059 MAP kinase inhibitor in AR4-2J cells. Total RNA was prepared from AR4-2J cells treated with TNF-α in association with (B) 2 (AdvIκBα or PDTC) NF-κB translocation inhibitors or (C) the MAP kinase inhibitors PD98059, SB202190, or SB A semiquantitative RT-PCR was performed using specific primers for rat PAP I or β-actin and the Access RT-PCR System kit (see Materials and Methods). Aliquots (5 μL) of the RT-PCR reactions were subjected to electrophoresis in 1.5% agarose gel and visualized by ethidium bromide staining.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Intracellular pathways of apoptosis regulation by TNF-α in AR4-2J cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions