Oxidative stress is elevated as a consequence of NPI-0052 and vorinostat interactions. (A) NAC partially protects from NPI-0052/vorinostat-induced apoptosis. Jurkat cells were pretreated with 24 mM NAC for 30 minutes, followed by treatment with diluent, 500 nM vorinostat, 5 nM NPI-0052, or combination of drugs for 24 hours. Apoptosis was assessed by PI staining and subsequent flow cytometric analysis. Statistical differences were calculated: *P < .05 for NPI-0052/vorinostat-treated cells compared with control (dimethyl sulfoxide [DMSO]); **P < .01 for NPI-0052/vorinostat compared with NPI-0052/vorinostat/NAC. (B) Intracellular superoxide production in Jurkat cells treated with NPI-0052/vorinostat regimen is decreased with NAC. Cells were treated as indicated for 12 hours, followed by staining with HEt and analyzed by flow cytometry. Shown are the mean fluorescence values of 3 independent experiments. (C) NPI-0052/vorinostat increases superoxide levels, which are decreased by NAC. Shown is a representative histogram of the 3 experiments performed for HEt staining (Figure 4B). Solid line represents control cells, solid bold line is 5 nM NPI-0052/500 nM vorinostat-treated cells and dashed line indicates 5 nM NPI-0052/500 nM vorinostat cells pretreated with 24 mM NAC. (D) NPI-0052 generates more superoxide levels than bortezomib in Jurkat cells. Cells were exposed to 5 nM NPI-0052, bortezomib, or diluent for 12 hours and stained with HEt. Samples were analyzed by flow cytometry. Shown are mean fluorescence values of 3 independent experiments.