Disruption of mitochondrial transmembrane potential (ΔΨm), cytochrome c release, and caspase-3 activation. (A) The figure shows the changes in ΔΨm in U-937 cells treated for 14 hours with 4 μMAs₂O₃ and LY294002, alone or in combination, as determined by flow cytometry after cell loading with R123. The vertical, dotted lines represent the mean fluorescence value in untreated cells (Cont), to better discern the displacement caused by the treatments. (B) The blot shows the presence of cytochrome c (Cyt c) in cytosolic extracts (25 μg protein per lane) obtained from untreated cells (Cont), from cells treated for 14 hours with LY291002 alone, and from cells treated for the indicated time periods with 4 μMAs₂O₃, alone or in combination with LY294002. The level of α-tubulin (α-tub) was also measured as a control. (C) The figure shows the relative caspase-3 activity, as determined using Ac-DEVD-pNA as substrate, in extracts from untreated cells (Cont) and cells treated for 24 hours with 4 μMAs₂O₃, 20 nM camptothecin, and 4 μM cisplatin, alone (-)orin combination with LY294002 (+LY). The results (mean ± standard deviation of 3 determinations) are represented in relation to the control, which was given the arbitrary value of one. All other conditions were as in Figure 1.