Figure 7. Active caspase-3 ELISA analysis of possible apoptosis in HLE-B3 cells treated with SB216763, UO126, staurosporine, or DMSO. The possibility of the onset of apoptosis was determined using an active capase-3 ELISA with HLE-B3 cells treated with SB216763; 12 µM), UO126; 10 µM), staurosporine (100 nM), or 0.05% DMSO vehicle. Treated and mock-treated HLE-B3 cells were incubated with serum-free minimal essential media (MEM) for 90 min in atmospheric oxygen. The cells were then switched to hypoxia for 180 min in the continued presence of each individual treatment. At the end of the hypoxic exposure, the media were removed and replaced with fresh, oxygenated media still containing SB216763, UO126, staurosporine, or DMSO vehicle. The cells were placed in atmospheric oxygen for 60 min and subsequently lysed, and the quantity of protein determined per treatment. Caspase-3 activity was determined using 10 µg of protein following the manufacturer’s instructions. Data are based upon results from three independent cell populations and were analyzed using GraphPad Prism 5. The error bars represent the standard error. Only treatment with staurosporine indicated a marked increase in activation of caspase-3.