FIGURE 4.

Down-regulation of Puma by shRNA protects cortical neurons from death. A, primary cultured rat cortical neurons (5 days in vitro) were transfected with pSIREN-shPuma-zsgreen or control pSIREN-shRand-zsgreen (scrambled shRNA) and maintained for 48 h and then subjected to Aβ (1.5 μm) treatment for 72 h. Representative pictures of transfected neurons that were maintained in the presence or absence of Aβ for the indicated time periods are shown. Images were taken under a ×20 objective. B, graphical representation of the percentage of viable green cells after each time point. The numbers of surviving transfected (green) cells were counted under a fluorescence microscope just before Aβ treatment (C) and after 24, 48, and 72 h of the same treatment. Data are from three independent experiments, each with comparable results, and are shown as mean ± S.E., performed in triplicates. The asterisks denote statistically significant differences from control (shRand) at corresponding time points: *, p < 0.05; **, p < 0.001. C, Puma knockdown prevents neuronal degeneration. Sholl analysis of single imaged neurons by using ImageJ was done as described under “Experimental Procedures.” Data represent the mean ± S.E. (error bars) of six different neurons from three independent cultures for each class. *, statistically significant differences from shRand (control); p < 0.001.