1. by Fawzi Aoudjit, and Kristiina Vuori
Engagement of the α2β1 integrin inhibits Fas ligand expression and activation-induced cell death in T cells in a focal adhesion kinase-dependent manner
by Fawzi Aoudjit, and Kristiina Vuori
Blood
Volume 95(6):
March 15, 2000
©2000 by American Society of Hematology

2. β1 integrin signaling inhibits AICD in Jurkat cells
β1 integrin signaling inhibits AICD in Jurkat cells.Jurkat cells were stimulated with or without 50 μg/mL of immobilized anti-CD3 antibody for 24 hours in the presence or absence of 50 μg/mL of immobilized anti-β1 or IgG control antibodies.
β1 integrin signaling inhibits AICD in Jurkat cells.Jurkat cells were stimulated with or without 50 μg/mL of immobilized anti-CD3 antibody for 24 hours in the presence or absence of 50 μg/mL of immobilized anti-β1 or IgG control antibodies. Apoptosis was determined by DNA fragmentation analysis as described in “Materials and methods.” The results are mean of 3 independent experiments, each done in a duplicate. *P < .05 between anti-CD3-stimulated control and anti-β1 antibody samples.
Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:
©2000 by American Society of Hematology

3. Collagen type I inhibits AICD in Jurkat cells
Collagen type I inhibits AICD in Jurkat cells.(A) Jurkat cells were stimulated with or without immobilized anti-CD3 antibody for 24 hours in the presence or absence of 50 μg/mL of the indicated ECM proteins.
Collagen type I inhibits AICD in Jurkat cells.(A) Jurkat cells were stimulated with or without immobilized anti-CD3 antibody for 24 hours in the presence or absence of 50 μg/mL of the indicated ECM proteins. Apoptosis was determined by DNA fragmentation analysis. Fn, fibronectin; Ln, laminin; Coll, collagen I. *P < .05 between anti-CD3-stimulated control and collagen-treated samples. (B) Cells were stimulated with or without PMA/ionomycin for 24 hours in the presence or absence of 50 μg/mL of collagen I (Coll I) or poly-l-lysine (PLL). Cell death was determined by propidium iodide uptake as described in “Materials and methods.” *P < .05 between PMA/ionomycin-stimulated control and collagen-treated samples. (C) Dose-response effect of collagen I on AICD. The results are representative of 3 independent experiments.
Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:
©2000 by American Society of Hematology

4. Collagen I-induced inhibition of AICD in Jurkat cells is mediated by the 2β1 integrin.(A) Jurkat cells were stimulated with or without anti-CD3 and collagen I in the presence or absence of inhibitory antibodies against α1 and α2 integrin subunits or contro...
Collagen I-induced inhibition of AICD in Jurkat cells is mediated by the 2β1 integrin.(A) Jurkat cells were stimulated with or without anti-CD3 and collagen I in the presence or absence of inhibitory antibodies against α1 and α2 integrin subunits or control antibodies (IgG). The anti-integrin antibodies were used at 25 μg/mL and were added 30 minutes before plating the cells on anti-CD3–coated wells. The cells were then cultured for 24 hours and cell death was determined by DNA fragmentation assay. (B) Adhesion assay of Jurkat cells on immobilized collagen I. Cells were activated or not with PMA/ionomycin and plated on wells that had been coated with collagen I and were allowed to attach for 1 hour at 37°C. 25 μg/mL of inhibitory anti-α1 and anti-α2 integrin antibodies were added to the wells as indicated in the figure. Adhesion was quantified as described in “Materials and methods.” The results are representative of 3 different experiments performed in triplicates.
Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:
©2000 by American Society of Hematology

5. Effect of 2β1 antibody cross-linking on AICD
Effect of 2β1 antibody cross-linking on AICD.Jurkat cells were cultured for 24 hours in wells that had been coated with anti-CD3 and with 50 μg/mL of activating anti-α2β1 antibody JSB2 or with control antibodies (IgG). 50 μg/mL of poly-l-lysine or collagen...
Effect of 2β1 antibody cross-linking on AICD.Jurkat cells were cultured for 24 hours in wells that had been coated with anti-CD3 and with 50 μg/mL of activating anti-α2β1 antibody JSB2 or with control antibodies (IgG). 50 μg/mL of poly-l-lysine or collagen I was added to some of the wells as indicated in the figure. DNA fragmentation was determined as previously described.
Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:
©2000 by American Society of Hematology

6. Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:2044-2051
©2000 by American Society of Hematology

7. Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:2044-2051
©2000 by American Society of Hematology

8. FACS analysis of Fas antigen expression on the surface of Jurkat cells
FACS analysis of Fas antigen expression on the surface of Jurkat cells.Jurkat cells were treated for 8 hours with PMA/ionomycin in the presence or absence of 100 μg/mL of poly-l-lysine or collagen I. The cells were harvested and stained as described in “Met...
FACS analysis of Fas antigen expression on the surface of Jurkat cells.Jurkat cells were treated for 8 hours with PMA/ionomycin in the presence or absence of 100 μg/mL of poly-l-lysine or collagen I. The cells were harvested and stained as described in “Methods.” The upper left panel represents negative and positive staining of unstimulated Jurkat cells with an isotype-matched control antibody and anti-Fas antibody, respectively. The 3 other panels show antiFas staining in stimulated Jurkat cells that had been treated with PMA/ ionomycin, poly-l-lysine and collagen I as indicated. X axis, relative fluorescence intensity; Y axis, cell number.
Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:
©2000 by American Society of Hematology

9. Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:2044-2051
©2000 by American Society of Hematology

10. Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:2044-2051
©2000 by American Society of Hematology

11. FAK is phosphorylated in response to 2β1 ligation and mediates 2β1-induced inhibition of AICD and Fas-L expression.(A) The cells were stimulated or not with collagen I at 20 and 50 μg/mL either alone or in combination with 20 μg/mL of soluble anti-CD3 for...
FAK is phosphorylated in response to 2β1 ligation and mediates 2β1-induced inhibition of AICD and Fas-L expression.(A) The cells were stimulated or not with collagen I at 20 and 50 μg/mL either alone or in combination with 20 μg/mL of soluble anti-CD3 for 10 minutes. The cells were washed and lysed and subjected to immunoprecipitation with anti-FAK antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine antibodies (top panel) and with antibodies against FAK (lower panel). (B) Jurkat cells were cotransfected with a plasmid encoding the dominant-negative form of FAK (FRNK), wild-type form of FAK (FAK), combination of FRNK- and FAK-encoding plasmids, or a control plasmid, together with a plasmid encoding GFP as described in “Materials and methods.” Viable cells were recovered 48 hours later by Ficoll gradient and stimulated with or without PMA and ionomycin in the presence or absence of 50 μg/mL collagen I for 24 hours. The cells were then washed and propidium iodide was added for 15 minutes on ice, and apoptosis was analyzed by FACScan®. The analysis was carried out on the double positive cell population for propidium iodide and fluorescent GFP. (C) Jurkat cells were cotransfected with plasmids encoding FRNK, FAK, Fas-L promoter reporter construct, and β-galactosidase. After 48 hours, the cells were washed and treated with the various stimuli as in (B) and luciferase activity (RLU) was determined after 18 hours.
Fawzi Aoudjit, and Kristiina Vuori Blood 2000;95:
©2000 by American Society of Hematology