An increase in [Ca
is necessary for Khz-cp-induced ROS generation and apoptosis. (A) SNU-1 cells were loaded with fura-2 AM for 30 min, and changes in [Ca2+]
after Khz-cp treatment (50 μg/ml) were analyzed by digital imaging microscopy. (B) SNU-1 cells were pretreated with EGTA (10 μM) or BAPTA-AM (10 μM) for 30 min, and membrane fractions were prepared for immunoblot analysis 12 h after Khz-cp treatment. (C-E) Cells were pretreated with EGTA or BAPTA-AM for 30 min and treated with Khz-cp (1:100 dilution). After 12 h, ROS generation was measured in SNU-1 cells by an Amplex Red hydrogen peroxide assay. The data are presented as the mean ± SD values (C). Cytosolic cytochrome c levels were analyzed in SNU-1 cells by immunoblot analysis (D). Apoptosis was analyzed 12 h after Khz-cp treatment (E).