Acetylation of p53 (e.g. sham), Dunnett’s test was adopted for post-hoc analyses after ANOVA. Statistical significance was accepted at the 95% confidence level (p,0.05). Data was expressed as mean6standard error (SE). Results 17b2E2 Attenuates Cerebral Ischemia-Induced Elevation of p53 and Acetyl p53-Lysine373 Levels in the Hippocampal CA1 Region Figure 1A shows that p53 levels in the hippocampal CA1 region are significantly increased at 6 h and 1 d after cerebral ischemia (Pla) as compared to sham controls. P53 levels at 3 h after reperfusion showed a pattern for elevation, which, however, was not statistically significant. In addition, 17b2E2 treatment significantly attenuated p53 elevation at 3 h, 6 h and 24 h after ischemic reperfusion (Fig. 1A). Figure 1B shows Acetyl p53-Lysine373 levels (expressed as ratio of Acetyl p53-Lysine373/ p53) in the hippocampal CA1 region at various time-points after GCI. The data shows that Acetyl p53-Lysine373 levels increase significantly in the hippocampal CA1 region at all time-points examined after GCI (3 h, 6 h and 24 h) as compared to sham controls. In addition, 17b2E2 treatment significantly attenuated ischemia-induced Acetyl p53-Lysine373 levels at all time-points after GCI reperfusion, with the most robust effect seen at 3 h following ischemic reperfusion (Fig. 1B). To determine in which cell types acetylation of p53 occurred, we performed double immunohistochemistry on hippocampal CA1 sections collected at 24 h after GCI for the neuronal marker, NeuN (red), and Acetyl p53-Lysine373 (green) (Fig. 1C). The results show that Acetyl p53- Lysine373 staining is highly co-localized in neurons, and confirm the ischemic reperfusion elevation of Acetyl p53-Lysine373 levels in the CA1 region after cerebral ischemia (Pla vs. Sham), and the Figure 1. P53 levels and acetylation of p53 on Lysine373 are increased in the hippocampus following global cerebral ischemia and attenuated by 17b-estradiol. (A & B) Homogenates obtained from the hippocampus CA1 at 3 h, 6 h and 24 h after reperfusion were subjected to Western blot analyses to examine the temporal expression of p53 (A) and Acetyl p53-Lys373 (B). Actin bands represent loading control. Data is expressed as fold differences as compared to sham (A) and Acetyl p53/p53 protein ratio expressed in relative units (B) from four to five animals. *p,0.05 vs. Sham, #p,0.05 vs. Pla control; S = Sham, P = Pla, E = E2. (C) Representative hippocampal CA1 sections were double labeled with the neuronal marker NeuN (red) and Acetyl p53 (green) at 24 h following ischemic reperfusion. Merged images represent acetylated p53 in pyramidal CA1 neurons of the hippocampus. Magnification 40X. n = 4–5 animals per treatment group. doi:10.1371/journal.pone.0027039.g001 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e27039