Fig. 3

Induction of Fas-L expression by both VEINCTR-N and sFas. (A) Cytofluorimetric measurement of membrane-bound Fas-L in Jurkat, WC8, and WR19L cells after treatment with those inhibitors. The cells were incubated in the presence of EDTA to prevent shedding of the ligand by metalloproteinases. Both Jurkat and WC8 almost doubled their Fas-ligand (Fas-L)-positive subsets; whereas, the control Fas-negative WR19L cells were not influenced. (B) (left graphic) Detection of Fas-L release in supernatants from Jurkat, WC8, and WR19L cells incubated overnight with 5 µg/ml of soluble Fas (sFas). A significant increase of sFas-L was revealed in culture media from both Jurkat and WC8, compared with untreated control cells (p < 0.02), in contrast with the defective response of WR19L cells. As expected, parallel control cell preparations stimulated by sFas in the presence of EDTA maintained a low level of Fas-L shedding. (right graphic) Measurement of Fas-L mRNA by RT-PCR in Jurkat cells treated with CH11, sFas, and VEINCTR-N. Analysis by the Fluor-S MultiImager gel analyzer confirmed a variable, though significant, Fas-L mRNA increment (p < 0.03) in response to the overnight incubation with sFas, VEINCTR-N, or CH11, compared with untreated control cells. By contrast, no difference of β-actin mRNA expression was observed. O.D., optical density.