E2F1/CDK1 expression is necessary for trophic deprivation-induced neuronal apoptosis.

<p>Rat cortical neurons were co-transfected with expression plasmids for ß-galactosidase with, either empty vector or vector expressing E2F1 or CDK1 (A and B). Similarly, ß-galactosidase plasmid was co-transfected along with scrambled, E2F1 or CDK1 shRNAs (C, D and E) and the extent of apoptosis was examined 48 h after transfection, or after an additional 24 h of trophic deprivation (TD) induction post 48 h transfection. <b>A</b>. Neurons transfected with E2F1 vector (0.6 µg DNA/0.5×10⁶ neurons) increased basal apoptosis as compared to empty vector. <b>B</b>. Neurons transfected with CDK1 (0.8 µg DNA/0.5×10⁶ neurons) vector enhanced TD-induced apoptosis as compared to empty vector transfected cells. <b>C–D</b>. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons transfected with two different shRNAs targeting either E2F1 or CDK1. <b>E</b>. Attenuation of TD-induced chromatin condensation in neurons transfected by CDK1 shRNA1 is shown. Representative photomicrographs of the shRNA transfected neurons, which also co-express GFP protein from the separate promoter are shown. In all experiments ß-galactosidase was co-transfected at 0.01 µg DNA/0.5×10⁶ neurons to visualize transfected neurons at later apoptotic stage. Neurons with either normal diffuse chromatin morphology or apoptotic condensation of nuclei (after Hoechst 33258 chromatin staining) are indicated by arrowheads or arrows, respectively. Statistical analysis was performed by Kruskal-Wallis one-way ANOVA on ranks, followed by post hoc adjustments using Dunnett's test. For A ^*p<0.001, vs. vector; For C ^*p<0.01, vs. TD vector; For D ^*p<0.05, vs. TD vector; Error bars are ± S.E.M. from three independent experiments.