Figure 3.
Suppression of endogenous p53 confers robust neuroprotection in hippocampal neurons. A, Top left, Schematic drawing of rAAV vector for shRNA expression. Bottom left, Immunoblot analysis of p53 expression in hippocampal neurons infected with rAAV–control--RNAi or rAAV–trp53–RNAi. Tubulin was used as loading control. Right, Immunostaining analysis (using antibodies to p53) in hippocampal neurons infected with rAAV–control--RNAi or rAAV–trp53--RNAi. Photomicrographs of p53 immunoreactivity, mCherry fluorescence, and Hoechst staining are shown. Scale bar, 20 μm. B, QRT-PCR analysis of trp53, apaf1, and bbc3 expression in uninfected hippocampal neurons and in hippocampal neurons infected with rAAV–control–RNAi or rAAV–trp53–RNAi. Data represent means ± SEM (n = 4). Statistically significant differences were analyzed by ANOVA; ***p < 0.001; NS, not significant. C, Analysis of apoptosis after STS treatment or growth factor withdrawal (−GF) in uninfected hippocampal neurons and in hippocampal neurons infected with rAAV–control–RNAi or rAAV–trp53–RNAi with or without a 16 h period of AP bursting induced by bicuculline (50 μm) before STS treatment or GF withdrawal. Bars represent means ± SEM (n = 4). Statistical analysis was determined by ANOVA; ***p < 0.001; NS, not significant.