American Society of Hematology

Figure 5.

$Figure 5. Disruption of rafts and absence of the first 40 intracellular amino acids lead to dislocation of FasL from rafts into disordered membrane fractions and to diminished FasL-induced cell death. (A) Dynamic confinement of GFP-FasL into discrete domains at the plasma membrane is independently prevented by enzymatic modification of cholesterol and deletion of the first 40 intracellular FasL amino acids. COS-7 cells transiently expressing the recombinant GFP-FasL protein were left untreated (▪) or were treated with cholesterol oxidase (□). Alternatively, COS-7 cells were transfected with GFP-FasL delta 1-40 (▵). FCS measurements were performed at 37°C immediately after cellular treatment. (B) Jurkat cells stably transfected with human FasL (hFasL) or hFasL delta 1-40 were solubilized in Brij 98 detergent and subjected to sucrose gradient separation. Immunoblots were performed on pooled heavy (8-9; HF) and light (1-4; Raft) fractions with the indicated antibodies. (C) HEK293 cells stably transfected with human FasL were treated with cholesterol oxidase (2 U/mL) for 2 hours (CO) or were left untreated (NT). Cells were then solubilized in Brij 98 detergent and subjected to sucrose gradient separation before analysis of the raft and nonraft fractions by Western blot. (D) JH6.2 cells were cocultured for 8 or 20 hours with Jurkat cells stably transfected with hFasL or hFasL delta 1-40 or with mock-transfected Jurkat cells. Cell death was then quantified by flow cytometry analysis of propidium iodide–stained ethanol-fixed cells. The graph represents the average of 3 independent experiments, with error bars indicating the SD. Equal cell surface expression of FasL and FasL delta 1-40 in the Jurkat cell clones was demonstrated by flow cytometry analysis (bottom panel). The thin line indicates secondary antibody alone; thick line, Anti-FasL antibody (Nok-1) plus secondary antibody. (E) Aliquots of the FasL-transfected 293 cells (left untreated or treated with 2 U/mL cholesterol oxidase from the experiment described in panel B) were used for a 5-hour coculture with Fas-expressing JH6.2 Jurkat target cells to quantify the killing capacity of FasL. As a control, JH6.2 cells were cocultured with mock-transfected HEK293 cells. The graph represents the average of 3 independent experiments, with error bars indicating SD. Flow cytometry analysis was performed with anti-FasL antibody (Nok-1) to ensure that cell surface expression of FasL was not modified by the cholesterol oxidase treatment (bottom panel). Overlay of profiles obtained for cells incubated with fluorescence-labeled secondary antibody alone or with Nok-1 plus secondary antibody are shown. The thin line indicates not treated (NT) cells; thick line, cholesterol oxidase (CO)–treated cells.$

Disruption of rafts and absence of the first 40 intracellular amino acids lead to dislocation of FasL from rafts into disordered membrane fractions and to diminished FasL-induced cell death. (A) Dynamic confinement of GFP-FasL into discrete domains at the plasma membrane is independently prevented by enzymatic modification of cholesterol and deletion of the first 40 intracellular FasL amino acids. COS-7 cells transiently expressing the recombinant GFP-FasL protein were left untreated (▪) or were treated with cholesterol oxidase (□). Alternatively, COS-7 cells were transfected with GFP-FasL delta 1-40 (▵). FCS measurements were performed at 37°C immediately after cellular treatment. (B) Jurkat cells stably transfected with human FasL (hFasL) or hFasL delta 1-40 were solubilized in Brij 98 detergent and subjected to sucrose gradient separation. Immunoblots were performed on pooled heavy (8-9; HF) and light (1-4; Raft) fractions with the indicated antibodies. (C) HEK293 cells stably transfected with human FasL were treated with cholesterol oxidase (2 U/mL) for 2 hours (CO) or were left untreated (NT). Cells were then solubilized in Brij 98 detergent and subjected to sucrose gradient separation before analysis of the raft and nonraft fractions by Western blot. (D) JH6.2 cells were cocultured for 8 or 20 hours with Jurkat cells stably transfected with hFasL or hFasL delta 1-40 or with mock-transfected Jurkat cells. Cell death was then quantified by flow cytometry analysis of propidium iodide–stained ethanol-fixed cells. The graph represents the average of 3 independent experiments, with error bars indicating the SD. Equal cell surface expression of FasL and FasL delta 1-40 in the Jurkat cell clones was demonstrated by flow cytometry analysis (bottom panel). The thin line indicates secondary antibody alone; thick line, Anti-FasL antibody (Nok-1) plus secondary antibody. (E) Aliquots of the FasL-transfected 293 cells (left untreated or treated with 2 U/mL cholesterol oxidase from the experiment described in panel B) were used for a 5-hour coculture with Fas-expressing JH6.2 Jurkat target cells to quantify the killing capacity of FasL. As a control, JH6.2 cells were cocultured with mock-transfected HEK293 cells. The graph represents the average of 3 independent experiments, with error bars indicating SD. Flow cytometry analysis was performed with anti-FasL antibody (Nok-1) to ensure that cell surface expression of FasL was not modified by the cholesterol oxidase treatment (bottom panel). Overlay of profiles obtained for cells incubated with fluorescence-labeled secondary antibody alone or with Nok-1 plus secondary antibody are shown. The thin line indicates not treated (NT) cells; thick line, cholesterol oxidase (CO)–treated cells.

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