I3C inhibits the TNF-induced expression of NF-κB-dependent genes. (A) I3C inhibits the NF-κB-dependent reporter gene expression induced by TNF and TNFR1. A293 cells were transiently transfected with an NF-κB-containing plasmid alone or with TNFR1-expressing plasmids for 24 hours. After transfection, cells were washed and treated with the indicated concentrations of I3C for 24 hours. For TNF-treated cells, cells were washed and treated with 1 nM TNF for an additional 24 hours. The supernatants of the culture medium were assayed for SEAP activity as described in “Materials and methods.” Values represent the mean ± SD of triplicate cultures. (B) I3C inhibits TNF-induced cyclin D1, COX-2, and MMP-9 expression. Jurkat cells were incubated with 25 μM I3C and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against cyclin D1, COX-2, and MMP-9. (C) I3C inhibits the expression of TNF-induced antiapoptotic proteins. Jurkat cells were incubated with 25 μM I3C and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against survivin, IAP1, IAP2, XIAP, Bcl-2, Bfl-1/A1, TRAF1, FLIP, and β-actin.