Figure 5.

Intense proapoptotic stimuli do not kill Q97-expressing neurons by apoptosis. SCG neurons were infected with Ad-Q25 or Ad-Q97 or left uninfected. After 2 d, each type of culture was deprived of NGF and treated with 1 mm AraC (AraC/-N or A/-N) for 24 h. A, Apoptotic morphology: cells were fixed and stained with Hoechst 33342 to delineate nuclear morphology. Scale bar, 10 μm. B, Quantification of apoptosis (mean ± SD; n = 4; p < 0.001, Student's t test). C, Uninfected (Un), Q25-expressing, and Q97-expressing neurons express apoptotic signals in response to A/-N. Neurons were left untreated or treated from days 2 to 3 with 1 mm AraC in the absence of NGF (A/-N). BAF (50 μm) was added to all cultures to prevent loss of neurons undergoing apoptosis. Extracts were probed for P-ser15–p53, BimEL, and Hsp70, using tERK as loading control. D, A/-N induces Bax activation only in neurons devoid of IB. Neurons were infected with Ad-Q25, Ad-Q47, or Ad-Q97, and Bax activation was detected by immunostaining for active Bax with anti-Bax 1D1 (red); nuclei stained with Hoechst are in blue, and expression of Q25, Q47 (inset), and Q97 is indicated in green. Note normal Bax activation in Q47-expressing neurons. E, RT-PCR detection of Puma and Bim mRNA. Uninfected neurons or neurons expressing Q25 or Q97 for 2 d were left untreated or treated with 1 mm AraC in the absence of NGF but in the presence of 50 μm BAF for 12 h. The amount of mRNA was normalized to control GAPDH mRNA for each extract, setting the values from uninfected controls to 1 to calculate fold change. Mean ± SD; n = 3; p < 0.05, ANOVA; p < 0.05 versus control Tukey's honestly significant difference test except Q97/Puma, which was not significantly changed.