Fig. 1. Enhancement of TNF-R1-induced cell death by costimulation of TNF-R2, CD40 or CD30. (A) HeLa transfectants stably expressing TNF-R2, CD40 or CD30 were analyzed for expression of these receptors by FACS analysis with receptor-specific monoclonal antibodies. (B,C) HeLa-TNF-R2, -CD40 and -CD30 cells were grown in 96-well microtiter plates (15x10³ cells/well) and cultured overnight at 37°C. The next day the cells were treated overnight in triplicates with TNF (B), crosslinked FasL (C) or crosslinked TRAIL (D) in the presence of CHX (2.5 µg/ml) with ([UNK]) or without () costimulation of the respective TRAF2-interacting receptor. TNF-R2 was triggered with a polyclonal TNF-R2-specific IgG preparation (2 µg/ml), CD30 was stimulated with the agonistic mAb Ki-1 (3 µg/ml) and CD40 was activated with soluble Flag-tagged CD40L (100 ng/ml) crosslinked with the anti-Flag mAb M2 (1 µg/ml). Finally, viable cells were quantified by crystal violet staining.