Combined treatment of leukemia cells with adaphostin and MG-132 or bortezomib leads to an increase in ROS generation and lethality, events that are attenuated by the free-radical scavenger NAC. (A) Jurkat cells preteated with or without 6 mM NAC for 3 hours or 1000 U/mL catalase for 1 hour were exposed to 400 nM adaphostin plus or minus 200 nM MG-132 or 4.0 nM bortezomib (Btzmb), and ROS generation was monitored by flow cytometry using DHCF as the dye following 18 hours of drug exposure. (B) Jurkat cells were treated with 400 nM adaphostin plus or minus 200 nM MG-132, after which levels of ROS generation were measured at the indicated interval (eg, 0.5 to 24 hours). (C) Jurkat cells preteated with or without 6 mM NAC for 3 hours or 10 μM Boc-fmk for 30 minutes or 1000 U/mL catalase for 1 hour were exposed to 400 nM adaphostin plus or minus 200 nM MG-132 for 24 hours, after which apoptotic cells were monitored by annexin V/PI staining and flow cytometry as described previously. (D) Jurkat cells were treated with 400 nM adaphostin plus or minus 200 nM MG-132 for 16 hours or with 1 mM BSO for 24 hours. Then cells were harvested and homogenized. The samples were then deproteinated, and GSH level was determined as described in “Materials and methods.” Jurkat cells were (E) pretreated with or without 6 mM NAC for 3 hours or (F) pretreated with or without 10 μM Boc-fmk for 30 minutes followed by exposure to 400 nM adaphostin plus or minus 200 nM MG-132 for 8 hours. Following drug treatment, cytosolic (S-100) fractions and whole-cell lysates were obtained as described in “Materials and methods.” Protein samples were subjected to Western blot analysis using the indicated primary antibodies. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. ^*Significantly less than values for cells exposed to drugs in the absence of NAC or Boc-fmk; P < .005.