Pan-AKIs block TRAIL-induced canonical and noncanonical NF-κB activation in MM cells. (A) HMCLs were treated with pan-AKIs for 3 hours and then incubated with TRAIL (2.4 ng/mL in RPMI 8226 and OPM-2, 9.6 ng/mL in RPMI 8226/R5 and U266, 300 ng/mL in JJN3) and after 24 hours nuclear extracts were prepared. (i) Nuclear extracts from RPMI 8226 and RPMI 8226/R5 were immunoblotted against NF-κB p65, NF-κB1 p50, NF-κB2 p52, c-Rel, RelB, and laminin or histone H2B as nuclear loading control. Bands were subjected to densitometric scanning using the TINA 2 software, and the number below each lane represents the relative amount of the indicated proteins normalized to laminin or histone H2B expression. (ii) HMCL nuclear extracts were tested for DNA binding activity of the NF-κB p65, p50, p52, c-Rel, and RelB subunits using the TransAM NF-κB enzyme-linked immunosorbent assay kit. Results were normalized to the untreated control. Values represent mean ± SD of 3 separate experiments performed in triplicate. (B) HMCLs were cultured with pan-AKIs and/or TRAIL as previously described. Endogenous p-IKKα/β, p-NF-κB2 p100, p-IκB-α, IKKβ, and IKKα from whole cell lysates were revealed by western blot analysis. Anti-actin immunoblotting was performed as loading control. Bands were subjected to densitometric scanning and p-IKKα/β, p-NF-κB2 p100, and p-IκB-α protein expression levels were normalized to actin; the number below each lane represents the relative amount of the indicated proteins. (Ci-ii) HMCLs were electroporated with IκB-α or nontargeting siRNA (CONT), and 24 hours after siRNA transfection the cells were treated with MK-0457. After 24 hours of treatment, cytoplasmic and nuclear extracts were prepared. (i) Cytoplasmic cell lysates were immunoblotted against IκB-α and tubulin as a marker of cytoplasmic separation as well as loading control. (ii) Nuclear extracts were tested for DNA binding activity of the NF-κB p65 subunit using the TransAM NF-κB enzyme-linked immunosorbent assay kit. Results were normalized to the untreated control. Values represent mean ± SD of 3 separate experiments performed in triplicate. (iii) Transfection of IκB-α, but not the unrelated nonspecific control siRNA, led to a decrease in IκB-α protein expression in HMCLs without affecting the levels of the unrelated protein actin. At 24 hours after siRNA transfection, HMCLs were treated with pan-AKIs for 3 hours and then incubated with TRAIL (2.4 ng/mL in RPMI 8226 and OPM-2, 9.6 ng/mL in RPMI 8226/R5 and U266, 300 ng/mL in JJN3) for 24 hours, and the percentages of annexin-V+ apoptotic cells were measured. Values are mean ± SD of 3 independent experiments (*P < .01 vs pan-AKIs/TRAIL nonspecific siRNA [CONT]; Dunnet test). (D) Transfection of Aurora A or B, but not the unrelated nonspecific control siRNA, led to a decrease in Aurora kinase protein expression in RPMI 8226 and RPMI 8226/R5 without affecting the levels of the unrelated protein actin. At 48 hours after siRNA transfection, cells were lysed for immunoblot analysis to monitor the expression of Aurora A and B, p-IKKβ, p-IKKα, p-IκB-α, p-NF-κB2 p100, IKKβ, IKKα, and actin as loading control. Bands were subjected to densitometric scanning and p-IKKα/β, p-NF-κB2 p100, and p-IκB-α protein expression levels were normalized to actin. The number below each lane represents the relative amount of the indicated proteins.