NPI-0052 inhibits TNF-induced NF-κB–dependent reporter gene (SEAP) expression and suppresses chymotrypsin-like activity of 20S proteasome. (A) A293 cells were transiently transfected with an NF-κB–containing plasmid linked to the SEAP gene and then pretreated with the indicated concentrations of NPI-0052 for 4 hours. After 24 hours in culture with 1 nM TNF, cell supernatants were collected and assayed for SEAP activity as described in “Materials and methods.” Results are expressed as fold activity over the activity of the vector control. (B) Chemical structures of various proteasome inhibitors. (C) KBM-5 (1 × 10⁶) cells were incubated with 50 nM each of various proteasome inhibitors 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. (D) RPMI 8226 and PC-3 cells were treated with various concentrations of NPI-0052 and bortezomib for 1 hour, prepared the cell lysates, and assayed for the chymotrypsin-like activity of the 20S proteasome as described in “Materials and methods.” Results are presented as percentage inhibition compared with the chymotrypsin-like activity observed in untreated cells. Error bars represent SD of triplicate values.