Dual inhibition of IKKα and IKKβ significantly inhibits MM cell growth. (A) MM.1S cells were transfected with scrambled (■) or IKKα (●) shRNA. Both cells were then cultured in the presence (2.5, 5 μM) or absence of MLN120B for 48 hours, and viable cells were enumerated by trypan-blue exclusion. Percentage growth inhibition was calculated as follows: (cell number after 24 hours with MLN120B treatment/cell number without MLN120B treatment) × 100. *P < .01. (B) MM.1S cells were cultured with 17AAG (1 μM) for 4, 8, and 12 hours. Whole cell lysates were subjected to immunoblotting with anti-IKKα, -IKKβ, and -α-tubulin Abs. (C) MM.1S cells were cultured with 17AAG (1 μM) for 4 hours and 8 hours. mRNA level was examined using real-time quantitative PCR, normalized to expression of GAPDH. (D) MM.1S and RPMI8226 cells were cultured with 17AAG (0.25 and 0.5 μM) for 8 hours. Nuclear extracts were subjected to EMSA. Exposure time of autoradiography varied for each cell line. The intensity of the bands was digitalized by ImageJ software and indicated fold decrease relative to nontreated cells. (E) MM.1S and RPMI8226 cells were cultured with 17AAG (0.5 μM) or MLN120B (5 μM) for 8 hours. Nuclear extracts were subjected to EMSA. (F) MM.1S cells were cultured with 17AAG (250 and 500 nM) in the presence or absence of MLN120B (5 μM) for 8 hours. Nuclear extracts were subjected to EMSA. MM.1S (G) and INA6 (H) cells were cultured for 48 hours in BMSC-coated (□) or noncoated (■) wells in control media or MLN120B (5 μM), with or without 17AAG (62.5, 125, or 250 nM). DNA synthesis was assessed by [³H]-thymidine uptake; data represent mean ( ± SD) of quadruplicate cultures.