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.
