Effect of Bcl-2 on MRP8/MRP14-induced cell death. (A) Status of Bcl-2 expression. Cell lysates were prepared from Jurkat cells stably overexpressing either Bcl-2 or the empty vector control and subjected to immunoblotting with anti–Bcl-2 antibodies. (B) Effect of Bcl-2 on MRP8/MRP14-induced DNA fragmentation and phosphatidylserine exposure. Jurkat cells overexpressing Bcl-2 or the empty vector control were treated for 24 or 48 hours with MRP8/MRP14 (▪) or STS (⊡), or left untreated (□). Subsequently, DNA fragmentation was determined by flow cytometric measurement of hypodiploid DNA (left panel). Exposure of phosphatidylserine exposure was measured by flow cytometric detection of annexin-V–positive/PI-negative cells (right panel). (C) Effect of Bcl-2 on caspase activation. Jurkat cells overexpressing Bcl-2 or the empty vector control were treated for 24 hours with MRP8/MRP14 (▪) or STS (⊡), or left untreated (□). Caspase-3–like (left panel) and caspase-9–like (right panel) activities were measured using the fluorimetric substrates Ac-DEVD-AMC and Ac-LEHD-AMC, respectively. Caspase activity is indicated as delta F per microgram of protein. The data represent the means ± SEM from 4 independent experiments. Note that the relatively weak caspase activation in the STS-treated samples is due to the fact that caspase activation is maximal within 12 hours after STS treatment and declines thereafter. *Significant decreases of the apoptotic and caspase-activating effects of MRP8/MRP14 and STS by Bcl-2 overexpression compared with empty vector control cells (P < .05).