FIG. 7.

Mutations in the AP-1 site of the CD95L promoter destroy the inducibility of the promoter constructs, and induction is inhibited by dominant-negative c-Jun. (A) The AP-1 site in the basal promoter was mutated as indicated. To investigate the effect on the basal promoter, Hep3B cells were transfected with the −36/+100 (CD95L wild-type promoter [prom wt]) or the APX4 (mutated −36/+100 CD95L.luc, CD95L mutant promoter [prom mut]) constructs, respectively. To investigate the effect on the full-length promoter, Hep3B cells were transfected with the −1204/+100 (CD95L prom wt) or the APX4/−1204 (mutated −1204/+100 CD95L.luc, CD95L prom mut) constructs, respectively. Transfection efficiency was monitored by cotransfection of Renilla luciferase. Following transfection, cells were treated with 5-FU (100 μg/ml) for 48 h. Luciferase activity was measured and fold induction was calculated. One representative experiment out of five performed is shown. (B) Influence of dominant-negative c-jun (DN c-jun). Hep3B cells were transfected with the −36/+100, −36/+19, or pnull.luc constructs. Cells were either cotransfected with a control plasmid (c) or an expression construct for dominant-negative c-jun (+). As a control, c-jun was also cotransfected in one experiment. After completion of the transfection, cells were split and one-half was treated with 100 μg of 5-FU/ml and the other half was left untreated. Bars show fold induction calculated as follows: relative light units (treated cells)/relative light units (untreated cells). Relative luciferase units were normalized by Renilla luciferase activity using the dual luciferase system. Three independent experiments were performed, and one representative experiment is shown. The asterisks indicate that the absolute luciferase values in this experiment were approximately 75-fold higher than in experiments performed without the cotransfection of c-jun.