FL-Bid or a Bid mutant that can not be cleaved by caspase-8 potently induces cell death in cultured rat hippocampal neurons. (A)Western blot analysis of Bid overexpression in cultured rat hippocampal neurons. Cells were co-infected with the wild-type (wt) FL-Bid adenovirus and the rtTA containing virus. Expression of Bid was induced by 1 μg/ml doxycycline treatment for 12 or 24 h. Control cells were infected but not induced. (B)Quantification of cells showing nuclear condensation in response to an overexpression of wt FL-Bid or the Bid(D59A) mutant (mt). Cells were co-infected with the wt or mt FL-Bid adenovirus and the rtTA containing virus. After 48 h, expression of Bid was induced by the addition of 1 μM doxycycline for 12 or 24 h. Cells exhibiting nuclear condensation or nuclear fragmentation were counted in 4 – 5 randomly chosen subfields after staining with the chromatin-specific dye Hoechst 33528. Data are means ± SEM from n = 3 experiments. (C)AIF immunofluorescence analysis in non-induced and induced hippocampal neurons. Cells were co-infected with the wt FL-Bid adenovirus and the rtTA containing virus and induced for 12 h. Note the mitochondrial AIF immunofluorescence in non-induced cells sparing the nuclear region, and AIF translocation to the nucleus in the induced neuron cultures. Bar = 10 μm.