NPI-0052 inhibits both inducible and constitutive NF-κB activation in a dose- and time-dependent manner. (A) KBM-5 cells were incubated with the indicated concentrations of NPI-0052 for 4 hours and then exposed to 0.1 nM TNF for 30 minutes. The nuclear extracts were subjected to EMSA to evaluate NF-κB activation. (B) Cells were preincubated with 50 nM NPI-0052 for the indicated times, treated with 0.1 nM TNF for 30 minutes, and then subjected to EMSA to evaluate NF-κB activation. H1299 (C) or A293 (D) cells were pretreated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for 30 minutes. The nuclear extracts were then prepared and assayed for NF-κB by EMSA as described in “Materials and methods.” (E) U266 cells with constitutive NF-κB activation cells were incubated with (±) 50 nM NPI-0052 for 12 hours. Nuclear extracts were prepared and analyzed for NF-κB activation by EMSA.