NPI-0052 (N) and bortezomib (B) inhibit NF-κB function in WM cells. (A) BCWM.1 cells were cultured with either NPI-0052 (10 nM), bortezomib (10 nM), or the combination for 4 hours, and then TNF-α (10 ng/mL) was added for the last 20 minutes. NF-κBp65 transcription factor binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. Wild-type and mutant are wild-type and mutated consensus competitor oligonucleotides, respectively. All results represent means (± SD) of triplicate experiments. (B,C) BCWM.1 cells were cultured with either NPI-0052 (10 nM), bortezomib (10 nM), or the combination for 4 hours, and TNF-α (10 ng/mL) was added for the last 20 minutes. Cytoplasmic and nuclear extracts were subjected to Western blotting using anti–p-NF-κBp65, –NF-κBp50, –NF-κBp52 IkBα, -RelB, –p-IκB, -IκB, -nucleolin, and –α-tubulin antibodies. (D) BCWM.1 cells were cultured with NPI-0052 (10 nM) and bortezomib (10 nM) for 4 hours, or control medium, and TNF-α (10 ng/mL) was added for the last 20 minutes. Immunocytochemical analysis was assessed using anti–p-NF-κBp65 antibody. DAPI was used to stain nuclei.