Bcl-x_Lexpression suppresses activation of mitochondrial phospholipase A₂. Increasing concentrations (0.1-100 μM as indicated) of arachidonic acid induce phospholipase A₂ activation and enhance anti-IgM–(1 μg/mL) stimulated PLA₂ activation (A) as determined by the increased release (intracellular and extracellular) of [³H]arachidonic acid from cellular [³H]arachidonic acid–labeled phosphatidylcholine as described in “Materials and methods.” Due to the loss of [³H]arachidonic acid during the cellular fractionation process, anti-IgM–(1 μg/mL) stimulated mitochondrial phospholipase A₂ activation (B-C) was therefore determined by the measurement of decreased levels of [³H]arachidonic acid–labeled phosphatidylcholine in isolated mitochondria derived from wild-type (B) and Bcl-x_L (C) overexpressing WEHI-231 B cells. Data are representative of at least 3 independent experiments. (D) WEHI-231 B cells were cultured for 48 hours with medium or anti-IgM (10 μg/mL) in the presence and absence of oligomycin (OM, 8 ng/mL) or cyclosporin A (CSA, 1 nM), and percent of apoptotic cells was measured by PI staining of subdiploid DNA content as described in “Materials and methods.” (E) WEHI-231 B cells were cultured for 3 hours with medium or anti-IgM (10 μg/mL) in the presence and absence of oligomycin (OM, 8 ng/mL) or cyclosporin A (CSA, 1 nM), and isolated mitochondria were prepared. PLA₂ activity was assayed using the PLA₂ assay kit as described in “Materials and methods.” Data are presented as means ± SD in which n = 3 and from single experiments that are representative of at least 3 independent experiments.