Induction of apoptosis by NaAsO₂ in HRS cell lines.(A) L540 cells were incubated for 20 hours with 0, 20, or 200 μM NaAsO₂, as indicated. Cells were stained with annexin V–FITC and PI and analyzed by fluorescence-activated cell sorter (FACS) analysis. The percentage of annexin V⁺ cells relative to the untreated cells is indicated. (B) HDLM-2 and L540 cells were treated with 20 μM arsenite. At the indicated times whole cell lysates were prepared and analyzed for cleavage of caspase-3 by Western blotting. The full-length protein (open arrows) and the cleavage products (closed arrows) are indicated. (C) HDLM-2 cells were treated with 0 (▵), 1 (▪), or 2 (⋄) μM NaAsO₂. After 24, 48, and 72 hours, the fraction of annexin V–FITC⁺ cells was determined by FACS analysis. Error bars denote SDs. (D) Inhibition of NaAsO₂-induced apoptosis by blockage of caspase-8– or caspase-3–like activity. Prior to the incubation with 20 μM NaAsO₂, HDLM-2 cells were treated with various concentrations of the caspase-8 inhibitor z-IETD-fmk and/or the caspase-3 family inhibitor z-DEVD-fmk, or, as control (C), dimethyl sulfoxide (DMSO) alone. After 20 hours of treatment with NaAsO₂, the percentage of apoptotic cells was determined by acridine orange staining. Error bars denote SDs.