Figure 4.
Caspase-8 is dispensable for EtOH-induced apoptosis. WT and Casp8Δhepa mice were fed with isocaloric (ctrl) or Lieber-DeCarli (EtOH) diet for 8 weeks. (a) Analysis of cell death by TUNEL staining (top) and quantification (bottom). Apoptotic cells are stained in green (arrows); total cells were counter-stained with DAPI (blue). (b) Analysis of Caspase-3 activation. Top: liver cryosections were stained with an antibody specific for activated (i.e. cleaved; cl.) Caspase-3. Cytoplasmic, activated Caspase-3 is stained in green and highlighted with arrows; total nuclei are counter stained with DAPI (blue). Bottom: immunoblot analysis of cleaved (cl.) Caspase-3 in the liver of WT and Casp8Δhepa mice (bottom). Ratio: normalization of Caspase-3 expression was performed by densitometry. Values are given in arbitrary pixel units and were calculated as fold induction compared to WT (ctrl). (c) Determination of TNF gene expression by qPCR. (d) Expression and activation analysis of TNF-related immediate downstream factors STAT3, cJun and NF-κB by immunoblot from whole liver lysates. p-p65 membrane was over exposed (Long exp) to detect weak basal expression. Ratio: normalization of p65 expression by densitometry. (e) Expression analysis of Fas receptor (CD95/FasR) by qPCR analysis of mRNA expression (top), immunoblot analysis (middle) and immunofluorescence staining (bottom, red, arrows). **P<0.01; ***P<0.01; n.s.: not significant