TNF activates hepatocyte apoptosis in double-transgenic mice expressing ΔN-IκBα. (a) Caspase-3 activity. Single-transgenic (G) or double-transgenic (GN) mice were injected with 25 μg/kg TNF or saline 3 hours after injection with mifepristone or sesame oil. For comparison, C57BL6 wild-type (WT) mice were coinjected with TNF and D-galactosamine (GalN). Mice were killed 4 hours after TNF injection and liver protein lysates were used to measure caspase-3 activity by incubation with the fluorogenic substrate DEVD-AMC. Data represent average (± SEM) caspase-3 activity measured in each group. Three to five mice were used per group. *P < 0.05 compared with vehicle control. Inset: active caspase-3 Western blot analysis of mifepristone- and TNF-treated mice. (b) TUNEL assay. Liver sections from a single-transgenic (left) and a double-transgenic (right) mouse treated with mifepristone and TNF were incubated with FITC-dUTP and terminal deoxynucleotidyl transferase. Original magnification, ×400. (c) iNOS Western blot. (d) DNA synthesis. Mice were injected with BrdU prior to sacrifice at the indicated times after treatment with mifepristone and TNF. Liver sections were stained with an antibody to BrdU as described in Methods, and positive nuclei were scored for 30 fields (×400) per section. Data represent average (± SEM) percentage of BrdU-positive hepatocyte nuclei per group. Three mice were used per group.