HNK induces apoptosis in MM cells. (A) MM.1S and RPMI 8226 cells were treated with 8 μg/mL HNK for 48 hours, and apoptosis was assessed using TUNEL assay. (B) Cleavage of caspases and PARP was determined by Western blotting of MM.1S whole-cell lysates after 10 μg/mL HNK treatment for 12 hours and 24 hours, with or without z-VAD-fmk (25 μM) preincubation for 1.5 hours. (C) MM.1S cells were treated with HNK or As₂O₃, with or without 25 μM z-VAD-fmk pretreatment for 1.5 hours. Activation of caspase 3 was determined by flow cytometry. (D) MM cells were treated with HNK or As₂O₃ for 24 hours, with or without z-VAD-fmk (25 μM, 100 μM) pretreatment for 1.5 hours. Induction of apoptosis and cytotoxicity was determined by flow cytometry after APO2.7 staining and trypan blue exclusion, respectively. Values represent the mean plus or minus SD for 3 independent experiments. (E) MM.1S cells were treated with HNK (10 μg/mL for 0, 4, 8 and 12 hours). Whole-cell lysates were subjected to Western blotting to assess the expression of Bcl-2 family proteins. (F) MM.1S cells were treated with HNK (10 μg/mL for 24 hours), with or without pretreatment by z-VAD-fmk. Proteins in cytosolic fraction were subjected to immunoblotting of AIF and Endo G.