Gossypin inhibited the TNF-induced expression of the NF-κB-dependent genes TNFR1, TRADD, TRAF2, NIK, IKK-β, TAK-1, and TAB-1. (A-C) A293 cells were transiently transfected with NF-κB-containing SEAP reporter gene plasmid, alone or with the indicated plasmids, for 24 hours. After transfection, cells were washed and treated with 50 μM gossypin for 2 hours. To evaluate the effects of TNF treatment, we treated cells with 0.1 nmol/L TNF for an additional 24 hours. The supernatants of the culture medium were assayed for SEAP activity, as described in “Materials and methods.”