Figure 4: Akt-mediated phosphorylation of H2A on T17 delays apoptotic death in neurons.

(a) PC12 cells and primary cultured hippocampal neurons were treated with H₂O₂ (1 mM and 0.5 mM respectively) and cell extracts were immunoblotted with the indicated antibodies. (b) PC12 cells and primary hippocampal neurons were treated with H₂O₂ (1 mM and 0.5 mM respectively) for 30, 60, and 120 min. Cleaved caspase-3 was detected at 120 min. Fixed cells were stained with Hoechst 33342 (blue) and anti-cleaved caspase-3 antibody (green). Figures on the right indicate apoptotic cells with condensed chromatin morphologies. (c) PC12 cells were transfected with pGE-sh-Akt and pGE-mock (control) for 24 h and then exposed to H₂O₂ (1 mM) for the indicated time, followed by immunoblotting and quantitative analysis of cell death was determined by Hoechst staining. (d) PC12 cells, transfected with pGE-sh-Akt and pGE-mock (control), were infected with Adenovirus-GFP-Akt and then exposed to H₂O₂ (1 mM) for the indicated time, followed by immunoblotting and quantitative analysis of cell death was determined by Hoechst staining. (e) Primary hippocampal neurons were stained with anti-H2A-p17 antibody (red) after H₂O₂ exposure for the indicated time. Apoptotic cells measured by TUNEL assay are presented in the bar graph (bottom). *p < 0.05. (f) Primary hippocampal neurons were transfected with H2A-WT or H2A-T17A and exposed to H₂O₂. Fixed cells were stained with Hoechst 33342 (blue). Apoptotic cells with condensed chromatin morphologies measured by cell counting assay are presented in the graph. (g) PC12 cells were co-transfected with CA-Akt (or KD-Akt) and H2A-WT (or mutant construct), followed by H₂O₂ treatment for 120 min. Genomic DNA was separated on a 2% agarose gel. The percentage of cell death was determined at the indicated time point after H₂O₂ treatment. At least three separate experiments were performed and the results are the means of three independent experiments.