Combination treatment inhibits UPS and NF-κB pathway in MM cells. (A) MM cell lines were treated with FK866 (3 nM), bortezomib (2 nM), or combined therapy for 3 hours. Cells were then harvested, and intracellular NAD⁺ level was measured using an enzyme cyclic assay and normalized to total cell number. Data are mean ± SD of 2 independent experiments. (B) MM1S cells were treated with FK866 (3 nM) for 48 hours, and bortezomib (2 nM) was added for the last 6 hours. Cell extracts were then analyzed for 20S proteasome activities (CT-L, C-L, and T-L). Results are percentage inhibition of proteasome activities in drug in comparison with vehicle control–treated cells. Data are representative of 3 independent experiments. (C) MM1S and U266 cells were treated with FK866 (3 nM), bortezomib (2 nM), or combined therapy for 24 hours. Whole-cell lysates were then immunoblotted using antiubiquitin and antiactin Abs. Blots shown are representative of 3 independent experiments. (D) MM1S cells were treated with FK866 (3 nM), bortezomib (2 nM), or their combination for 24 hours. Cells were then fixed and stained with 4′,6-diamidino-2-phenylindole (blue) and antiubiquitin Ab. (E) MM1S cells were cultured with FK866 (3 nM), bortezomib (2 nM), or their combination for 6 hours, with TNF-α (10 ng/mL) added for the last 20 minutes. Cytoplasmic and nuclear extracts were subjected to western blotting using specific antibody for analysis of NF-κB canonical (anti–p-NF-κBp65 and -p-IκB) and noncanonical (-NF-κBp52, -RelB) activity. (F) MM1S cells were cultured with FK866 (3 nM), bortezomib (2 nM), or the combination for 6 hours, with TNF-α (10 ng/mL) added for the last 20 minutes. Immunocytochemical analysis was performed using anti-pospho-NF-κBp65 antibody. DAPI (4′,6-diamidino-2-phenylindole) was used to stain nuclei.