Bortezomib activates NF-κB in MM cell lines. (A) RPMI 8226 cells were cultured with increasing doses (10-40 nM) of bortezomib for 8 hours. (B) MM.1S, U266, OPM1, INA6, RPMI 8226, and OPM2 cells were treated with bortezomib (20 nM) for 8 hours. (C) MM.1S, U266, and RPMI 8226 cells were treated with bortezomib (10 nM) for indicated time periods. (D) RPMI 8226 cells were treated with bortezomib (20 nM) for 8 hours. Nuclear extracts were subjected to EMSA (A-C) or supershift assay (D) using anti-p65, (65) p50, (50) p52, (52) RelB (B), and cRel (cR) Abs. Oct-1 served as a loading control for EMSA. Exposure time of autoradiography varied for each cell line. (E) Alteration of gene expression after bortezomib treatment (20 nM, 6 hours) in RPMI 8226 cells. (F) RPMI8226 cells were cultured with DMSO control medium (□) or bortezomib (20 nM) for 1 hour (), 3 hours () and 6 hours (■). Gene expression of Bcl-xL, CD44, IRF4, and ICAM1 was analyzed by DNA microarray.