FIGURE 2.

Induction of apoptosis and IL8 production by GpL-TNFSF ligand fusion proteins and conventional soluble TNFSF ligands. A, HT1080 cells were grown in 96-well plates (20,000 cells per well) overnight, sensitized for apoptosis by treatment with 2.5 μg/ml cycloheximide and then challenged for an additional day with the indicated concentrations of GpL-FLAG-TNC-TRAIL and GpL-FLAG-TNC-TNF in the presence and absence of 1 μg/ml anti-FLAG mAb M2. Cell viability was finally quantified by crystal violet staining. B, HT1080 cells (for analysis of CD95L, TRAIL, LIGHT, LTα, and LTαβ₂) and HT1080 transfectants expressing 4-1BB, CD27, GITR, CD40, or OX40 were seeded in 96-well plates (10,000 cells per well). The next day, medium was changed to reduce the background of constitutively produced IL8, and cells were then challenged overnight in triplicate with increasing concentrations of the indicated TNFSF ligand variants in the absence and presence of 1 μg/ml of the FLAG-specific mAb M2. Finally, the IL8 content of supernatants was determined by ELISA analysis.