The CFZ/vorinostat regimen potently induces apoptosis in bortezomib-resistant SUDHL16-10BR, Raji 20-BR, and OCY-LY10-40BR cells. (A) OCI-LY10-40BR, Raji-20BR, and SUDHL16-10BR cells were treated with minimally toxic concentrations of CFZ and either vorinostat or SBHA for 48, 48, or 36 hours, respectively. Concentrations were as follows: (OCI-LY10-40BR) CFZ (20nM) with or without SBHA (40μM), or vorinostat (1.5μM); (Raji-20BR) CFZ (15nM) with or without SBHA (40μM) or vorinostat (2.0μM); (SUDHL16-10BR) CFZ (5nM) with or without SBHA (30μM) or vorinostat (1.25μM). Cell death was monitored by 7-AAD/DiOC₆ staining and flow cytometry. (B) Fractional effect values were determined for the combination treatments, after which median dose effect analysis was used to characterize the nature of the interactions. Combination index (CI) values less than 1.0 denote a synergistic interaction. (C) SUDHL16-10 BR cells were exposed to the indicated concentrations of CFZ and vorinostat as described in panel A for 24 hours, after which Western blot analysis was performed using the indicated antibodies. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. Each lane was loaded with 20 μg of protein. Two additional experiments yielded equivalent results. (D) SUDHL16-10BR cells were treated with the indicated concentrations of CFZ and vorinostat for 24 hours. Nuclear protein was extracted using the Nuclear Extract Kit (Active Motif), and NF-κB activity was determined using an ELISA TransAM NF-κB p65 Transcription Factor Assay Kit (Active Motif), as described in “NF-κB activity.” *Significantly less than values for cells treated with vorinostat alone (P < .002). In all cases, values represent the means for experiments performed in triplicate on 3 separate occasions plus or minus SD.