Increased Bak expression is required for bortezomib-induced apoptosis, but not death, of resistant cells. (A) siRNA-mediated knock-down of Bak, Bik, or Noxa was confirmed in whole cell lysates collected 24 hours following bortezomib treatment. (B) Knock-down of Bak significantly inhibited the ability of bortezomib to induce apoptosis in resistant cells (Raji 4RH shown here). Resistant cells transfected with siRNA specific for BAK1, BIK, or PMAIP1 (Noxa), or non-targeting siRNA (ctl) as a control, were treated with bortezomib (20nM) for 24 hours and assayed for caspase-3/7 activity. Data shown are from a representative experiment repeated at least 3 times. Bars indicate average caspase-3/7 activity from triplicate wells ± SD. Asterisks (*) denote a significant decrease in caspase-3/7 activity compared with ctl siRNA transfected cells within the same treatment group. (C) Knock-down of Bak (or Bik or Noxa) does not alter the ability of bortezomib to kill resistant cells (Raji 4RH shown here). Cell death was determined by propidium iodide uptake in the same population of cells from B following 72 hours of incubation with bortezomib (20nM). Data shown are the average of at least 3 independent experiments ± SD. No significant difference (P > .05) in the percentage of propidium iodide–positive (PI⁺) cells was seen comparing BAK1, BIK, or PMAIP1 (Noxa) siRNA transfected to control siRNA transfected cells within the same treatment group.