Coadministration of MLN4924 with belinostat disrupts NF-κB activation whereas Bim knockdown markedly attenuates apoptosis induced by this regimen. (A) U937 and MV-4-11 cells were exposed to PXD-101 (U937, 300 nM; MV-4-11, 200 nM) ± 100 nM MLN4924 for 24 hours, after which western blot analysis was performed using antibodies that specifically recognize neddylated (Nedd8) or acetylated (Ac) proteins. (B) U937 cells were incubated with 300 nM PXD-101 ± 100 nM MLN4924 for 16 hours, after which nuclear extracts were prepared and subjected to the TransAM NF-κB p65–DNA-binding enzyme-linked immunosorbent assay (ELISA) as per the manufacturer’s instruction. (C) Alternatively, total and S32/34 phosphorylated IκBα (top panels) as well as expression of NF-κB–dependent genes (bottom panels) were monitored by western blot analysis in U937 cells. (D) U937 and MV-4-11 cells were treated for 24 hours as described in panel A, after which western blot analysis was performed to monitor expression of the BH3-only proapoptotic protein Bim (including EL, L, and S isoforms). (E-F) U937 cells stably transfected with constructs encoding shRNA targeting human BCL2L11/Bim (shBim) or scrambled sequence as negative control (shNC) were treated with 300 nM PXD-101 ± 100 nM MLN4924 for 24 hours, after which western blot analysis and FCM were carried out to monitor Bim expression (E) and apoptosis (Annexin V, determined by flow cytometry) (F), respectively. For panels D and E, blots for BimEL were quantified using ImageJ software. Values indicate fold-increase of BimEL vs untreated control (arbitrarily set as 1.0), after normalization to β-actin or α-tubulin (α-tub). RLU, relative light unit; UT, untreated control.