Cotreatment of adaphostin with MG-132 synergistically induces cell death in human leukemia cells. (A) Jurkat cells were treated alone or in combination with the indicated concentrations of adaphostin and MG-132 (150 or 200 nM) for 24 hours, after which the percentage of apoptotic cells was monitored by annexin V/PI staining as described in “Materials and methods.” (B) Jurkat cells were treated with adaphostin (400 nM) or MG-132 (200 nM) individually as well as in combination, after which induction of apoptosis was monitored at intervals from 0 to 48 hours. (C) Jurkat cells were exposed to a range of adaphostin and MG-132 concentrations alone and in combination at a fixed ratio (eg, 2:1) simultaneously for 24 hours. At the end of this period, the percentage of cells undergoing apoptosis (reflected by annexin V/PI positivity) was determined for each condition. Fractional effect values were determined by comparing results with those of untreated controls, and median dose effect analysis was employed to characterize the nature of the interaction. Combination index values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. (D) U937 cells were treated with 750 nM adaphostin plus or minus 250 nM MG-132 for 24 hours. (E) HL-60 cells were treated with adaphostin (1.0 μM) plus or minus MG-132 (300 nM) for 48 hours. (F) Raji cells were treated with adaphostin (1.0 μM) plus or minus MG-132 (225 nM) for 36 hours, after which the percentage of apoptotic cells was monitored by annexin V/PI staining and flow cytometry. For panels A, B, D, E, and F, values represent the means plus or minus strandard deviation (SD) for 3 separate experiments performed in triplicate.