Figure 4: Hepatocyte caspase activity. Representative HPLC runs of liver tissue extracts showing intracellular caspase activity. Specimens were incubated at 37°C in 50 mL KH buffer (continuously gassed with 95% O2: 5% CO2) without additions or with the addition of 50 μM sorafenib or 50 μM regorafenib. At t =3 h, the specimens were transferred to the Ac-DEVD-AMC (casapse-3 substrate) reaction in the presence and absence of zVAD-fmk (pancaspase inhibitor). Tissues were then vigorously disrupted and the supernatants were separated on HPLC and analyzed for the AMC moiety (AMC retention time =4.6 min). Panel A: Intracellular caspase activity with and without zVAD-fmk at 0 h. Panel B: Intracellular caspase activity with and without zVAD-fmk at 3 h. Panel C: Intracellular caspase activity at 3 h in the presence of 50 μM sorafenib with and without zVAD-fmk. Panel D: Intracellular caspase activity at 3 h in the presence of 50 μM regorafenib with and without zVAD-fmk.