Figure 4.

Evidence for the activation of a mitochondrial death pathway in DS neurons. A, Left, The histogram shows the quantification of bax expression in NL (standardized as 100%) and DS cortical cultures at 8 DIV. Four normal and four DS independent cultures were used for the analysis. Values are expressed as mean ± SEM by unpaired Student's t test. ^*p < 0.05. Right, Representative Western blot showing bax expression in NL and DS neuronal cultures (anti-bax, 1:500). B, Differential interference contrast (DIC) imaging shows neurons with intact morphology in NL and DS cultures at 8 DIV. Double immunofluorescence of normal and DS neurons with anti-cytochrome c (1:500; cyt C) and anti-AIF (1:400; AIF) antibodies show mitochondrial localization of cytochrome c and AIF in cell bodies (NL; arrowheads) and processes (NL; arrows) of normal neurons. In contrast, DS neurons exhibit diffuse cytochrome c and AIF staining in both cell bodies (DS; arrowheads) and neuronal processes (DS; arrows). Bottom panels are higher magnifications of framed areas. Scale bar, 20 μm. C, Immunofluorescence shows positive immunoreactivity for active caspase 3 (1:1000) in DS neuronal cell bodies (red fluorescence; arrows). Normal neurons showed negative labeling for active caspase 3. Neurons were double labeled with anti-tubulin class III (1:2000; blue fluorescence). Scale bar, 20 μm.