3A). To determine whether C/EBPβ functioned to prevent RALA hepatocyte death from TNFα, the effect of C/EBPβ overexpression on TNFα-induced apoptosis in RALA hepatocytes Wnt inhibitor with an inhibition of NF-κB activation was assessed. Cells infected with the C/EBPβ-expressing

adenovirus WT-C/EBPβ alone or coinfected with WT-C/EBPβ and either Ad5LacZ or Ad5IκB expressed increased levels of C/EBPβ compared with cells infected with Ad5LacZ alone (Fig. 3B). Cells were coinfected with Ad5IκB and either Ad5LacZ as a control or WT-C/EBPβ and treated with TNFα. When compared with Ad5IκB/Ad5LacZ-coinfected cells, the amount of cell death after TNFα treatment was significantly decreased in Ad5IκB/WT-C/EBPβ–coinfected cells at 6 and 12 hours by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 3C). The ability of C/EBPβ expression to block cell death from TNFα was confirmed by fluorescence microscopic studies of Pexidartinib ic50 cells costained with acridine orange/ethidium bromide to quantify the numbers of apoptotic and necrotic cells. As previously established, death from NF-κB inactivation and TNFα was predominantly apoptotic, and no significant increase occurred in

the numbers of necrotic cells (data not shown). The marked increase in apoptotic cells with TNFα administration was significantly reduced by adenoviral expression of C/EBPβ (Fig. 3D). Thus, the NF-κB–dependent increase in C/EBPβ in TNFα-treated RALA hepatocytes is a mechanism

of cellular resistance to TNFα-induced apoptosis. The sensitization of hepatocytes to TNFα toxicity by NF-κB inhibition occurs through caspase-dependent apoptosis.17, 33 The ability of C/EBPβ to function as a caspase inhibitor suggested that the mechanism of C/EBPβ’s inhibition of TNFα-induced apoptosis may be through blocking caspase activation. Adenoviral expression of C/EBPβ significantly decreased levels of activity of the initiator caspase 8 in both untreated and TNFα-treated cells in which NF-κB was inhibited by Ad5IκB (Fig. 4A). Inhibition of caspase 8 by C/EBPβ prevented TNFα-induced activation of the mitochondrial death pathway as WT-C/EBPβ decreased 上海皓元 the amount of truncated Bid that translocated to the mitochondria and blocked the cytochrome c release from mitochondria into cytoplasm that occurred in Ad5IκB/Ad5LacZ-coinfected cells (Fig. 4B). In contrast, levels of cytochrome oxidase, a mitochondrial protein not released during apoptosis, were equivalent in Ad5LacZ- and WT-C/EBPβ–infected cells after TNFα treatment and indicated equal protein loading (Fig. 4B). As a result of the inhibition of cytochrome c release, downstream effector caspase 3 and caspase 7 activation was blocked in cells overexpressing C/EBPβ as detected by decreases in the active, cleaved caspase forms on immunoblots (Fig. 4C).