Defective TNF-α–mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice
J. Clin. Invest. Carmen García-Ruiz, et al. 111:197 doi:10.1172/JCI16010 [Go to this article.]

Figure 1
Mitochondrial GSH depletion facilitates TNF-α–mediated mitochondrial depolarization. (a) Cultured rat hepatocytes were pretreated with HP (1 mM) for 5 minutes and then fractionated to obtain cytosol and mitochondria for GSH determination by high-performance liquid chromatography. Results are expressed as means ± SD (n = 4 or 5). *P < 0.05 versus control. (b) After HP treatment, hepatocytes were then exposed to TNF-α (280 ng/ml) for 15–60 minutes. Peroxide formation and mitochondrial membrane potential were determined upon staining of cells with DFCDA and TMRM, respectively, and fluorescence of both fluorochromes was determined by flow cytometry. A representative profile of four independent experiments is shown. (c) Hepatocytes treated with TNF-α (280 ng/ml) over time with or without HP pretreatment were permeabilized with digitonin, and cell extracts were analyzed for cytochrome c or cytochrome c oxidase as indicated in the Methods. (d and e) Aliquots of cell extracts were used for the detection of active caspase 3 fragments by immunoblotting (d) or activity determination using a fluorescent peptide (e). Results are expressed as means ± SD (n = 3 or 4 independent experiments).