Fig. 4. Effect of pre- and costimulation of TNF-R2 on TNF-R1-mediated activation of NF-B and TNF-R1-induced cell death. (A-D) HeLa-TNF-R2 cells were grown in 96-well microtiter plates (15x10³ cells/well), cultured overnight at 37°C and were transiently transfected the next day with a plasmid mix containing 3xNF-B-luciferase reporter plasmid (20 ng/well), SV40 promoter-driven ß-galactosidase expression plasmid (10 ng/well) and empty vector (pCR3, 120 ng/well). 6 hours after transfection some cells (A,C) were stimulated overnight with 2 µg/ml anti-TNF-R2 IgG (filled bars) to allow next day analysis of TNF-R1-dependent NF-B activation in a situation where TRAF2 had already been depleted. For activation of TNF-R1, cells were challenged in triplicates with the indicated concentration of TNF (A) or the TNF-R1-specific agonistic mAb Htr1 (C). To analyze NF-B activation under costimulatory conditions (B,D), cells were stimulated with anti-TNF-R2 IgG (filled bars) and either TNF (B) or Htr1 (D). In all experiments, cells subjected to costimulation were compared with cells stimulated only with the respective concentration of TNF or Htr1 (open bars). After TNF-R1 stimulation for 6 hours, cells were lysed and assayed for luciferase and galactosidase activity. Finally, luciferase activities were normalized according to the respective galctosidase activities. (E) Effect of temporal order of TNF-R1 and TNF-R2 stimulation with respect to NF-B activation and apoptosis induction. TNF-R2 was stimulated for the indicated times before or after triggering of TNF-R1. For determination of NF-B activation ([UNK]), cells were transfected as described above and assayed for luciferase and galactosidase activities 6 hours after TNF-R1 triggering (10 ng/ml TNF). For determination of cell viability (), TNF-R1 was triggered overnight with 0.1 ng/ml TNF in the presence of CHX (2.5 µg/ml) (i.e. at a concentration of TNF that induces no cell death in the absence of TNF-R2 costimulation). The effect of TNF/anti-TNF-R2 IgG costimulation was normalized to the effect of TNF alone. (F) HeLa-TNF-R2 cells were seeded in 96-well microtiter plates at a density of 15x10³ cell/well and cultured overnight at 37°C in the absence () or presence ([UNK]) of anti-TNF-R2 IgG. The next day the cells were challenged with TNF (,) or TNF and anti-TNF-R2 IgG ([UNK]) in the presence of CHX (2.5 µg/ml). After overnight incubation, viable cells were quantified by crystal violet staining.