The LCL/LBH regimen diminishes activation of the noncanonical NF-κB pathway in MM cells. (A and B) U266 or MM.1S cells were exposed to the indicated concentrations of LCL ± LBH for 16 hours (U266) or 24 hours (MM.1S). Immunoblotting analysis was then performed to monitor levels of TRAF3, NIK, p52, TRAF2, and BCL-X_L. (C) U266/shNC and U266/shTRAF3 cells were exposed to the indicated concentrations of LCL ± LBH for 24 hours. Immunoblotting analysis was then performed to monitor levels of TRAF3, caspase-3, and PARP. (D) U266/EV and U266/NIK cells were exposed to the indicated concentrations of LCL (3 µM) ± LBH (7 nM) for 24 hours. Immunoblotting analysis was then performed to monitor levels of NIK, caspase-3, and PARP. DNA binding of NF-kB (p52 subunit) was determined by using a TransAM assay for NF-kB activity. (E and F) U266/GFP and U266/GFP-TRAF2 cells were exposed to the indicated concentrations of LCL ± LBH for 24 hours. Cells were stained with 7-AAD, and cytospin slides were prepared. Imaging was taken by fluorescence microscopy (bar = 40 µm). Immunoblotting analysis was then performed to monitor levels of GFP-TRAF2 and cleaved caspase-3. β-actin was assayed to ensure equivalent loading and transfer. All experiments were repeated three times CF, cleavage fragment; DAPI, 4′,6-diamidino-2-phenylindole.