JS-K induced cytotoxicity is mediated via NO^•. (A) MM.1S cells were cultured with 0 to 5 μM of JS-K (♦), 4-carbethoxy-piperazi/NO (○), N-carbethoxy-piperazine (▴), DNP-SG (▵), or CDNB (■) for 48 hours. Cell viability was assessed by MTT assay, and data represent means (± SD) of triplicate cultures. The final DMSO concentration in the cultures was 0.1% or less, which was found to be nontoxic (results not shown). (B) MM.1S cells were treated with JS-K (2.5 μM), and then intracellular NO^• was detected by flow cytometry using the NO^• indicator DAF-FM diacetate. The histogram plot shows the percentage of cells that have detectable levels of NO^• at 1, 2, and 4 hours. (C) MM.1S cells were cultured with JS-K (2.5 μM) for 4 hours after 1 hour of preincubation with GST inhibitors Cibacron Blue (20 μM), or sulfasalazine (50 μM). Percentage of NO^•-positive cells were then assayed by flow cytometry. Data represents mean (± SD) of triplicate experiments. *P < .01 compared with JS-K (2.5 μM)–only control. (D) MM.1S cells were cultured with JS-K (2.5 μM) for 24 hours after 2 hours of preincubation with NO^• scavengers cobalamin (50 μM) or NAC (100 μM). Cell death was assayed by flow cytometry after PI staining. Data represents mean (± SD) of triplicate cultures. *P < .01 compared with JS-K–only control.