FIGURE 4.

EtOH-induced cell death is inhibited with caspase-2 inhibitor. The cultured corneal fibroblasts were pretreated with or without caspase-2 inhibitor followed by EtOH (20%, 20 s) exposure. The inhibition of EtOH-induced caspase-2 substrate-cleavage activity with caspase-2 inhibitor (A) and the direct influence of caspase-2 inhibitor on caspase-3 cleavage activity (B) were determined. Cell viability (C) and apoptosis (D) was determined by CellTiter-Fluor Cell Viability assay and stained with annexin V-PI for flow-cytometric analysis, respectively. Data were derived from three independent experiments (means ± S.D.; *, p < 0.05 compared with the control group; #, p < 0.05 compared with the EtOH-treated group). E, loss of mitochondrial membrane potential was demonstrated by the change in JC-1 derived fluorescence from red (high potential as JC-1 aggregates) to green (low potential as JC-1 monomer). With or without caspase-2 inhibitor (20 μm), MPT was determined at 0.5, 1, 1.5, and 2 h. Data represent the results of three independent experiments performed in triplicate (means ± S.D.; *, p < 0.05 compared with the control group; †, p < 0.05 compared with the EtOH-treated group).