neuroprotective effect of 17b2E2 (data not shown). Our results are in agreement with the breast cancer literature, where 17b2E2 similarly was found to reduce p53 methylation at Lysine372 [51,52]. Our study also demonstrated the ability of 17b2E2 to regulate the p53-induced BH3 family members, Puma and Noxa. We showed that 17b2E2 neuroprotective properties prevented the elevation of these factors involved in the neuronal apoptotic pathway at 24 h after reperfusion. Work from transient GCI studies measuring Puma expression in the hippocampus CA1 [25] and in the cortex of MCAO ischemic animals [25] similarly confirms Puma elevation at 24 h following ischemic reperfusion, while the breast cancer literature provides support for 17b2E2 downregulation of Puma transcriptional activity [53]. Overex- pression of Puma has also been shown to be sufficient to induce Bax (Bcl-2-associated X protein)-dependent neuronal death in cultured neuronal cells, further supporting its pro-apoptotic role in neurons [17]. Our data also demonstrates that 17b2E2 treatment induces an increased interaction of p53 with the E3 ubiquitin ligase, Mdm2, which is correlated with enhanced ubiquitination and a decrease in p53 protein levels. Interestingly, p53 acetylation sites Lysine373 and Lysine382 have been shown to be the same sites for MDM2 binding [54,55]. Consequently, acetylation of p53 at these sites has been shown to result in an inability of MDM2 to bind p53 for degradation, and thus, p53 is kept at a high level [56]. In further support of this suggestion, administration of a histone acetylase inhibitor that specifically enhances acetylation at these sites has been shown to result in a significantly prolonged half-life of p53 by decreasing p53 ubiquitination [54]. Previous work has shown that the acetyltransferase, CBP/p300, plays a major role in acetylating p53. Our studies showed that administration of the CBP/p300 inhibitor, curcumin, was strongly neuroprotective against GCI, which is consistent with an important role for acetylated p53 in neuronal damage and cell death following cerebral ischemia. Finally, an additional interesting finding of our study was that the ability of 17b2E2 to attenuate p53 acetylation was lost if the 17b2E2 replacement was preceded by a period of long-term estrogen deprivation (LTED) (10 week ovariectomy). We previ- ously showed that the neuroprotective effect of 17b2E2 in GCI is similarly lost following LTED [36]. The loss of the ability of 17b2E2 to attenuate acetylation of p53 in the LTED animals could potentially explain the loss of the neuroprotective effect of 17b2E2 reported previously. An additional intriguing finding was References 1. Howard VJ, Kleindorfer DO, Judd SE, McClure LA, Safford MM, et al. (2011) Disparities in stroke incidence contributing to disparities in stroke mortality. Ann Neurol 69: 619–627. 2. George MG, Tong X, McGruder H, Yoon P, Rosamond W, et al. (2009) Paul Coverdell National Acute Stroke Registry Surveillance - four states, 2005-2007. MMWR Surveill Summ 58: 1–23. 3. MMWR (2007) Prevalence of stroke--United States, 2005. MMWR Morb Mortal Wkly Rep 56: 469–474. 4. Woodruff TM, Thundyil J, Tang SC, Sobey CG, Taylor SM, et al. (2011) Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol Neurodegener 6: 11. 5. Bacigaluppi M, Comi G, Hermann DM (2010) Animal models of ischemic stroke. Part two: modeling cerebral ischemia. Open Neurol J 4: 34–38. 6. Garcia-Yebenes I, Sobrado M, Zarruk JG, Castellanos M, Perez de la Ossa N, et al. (2011) A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke. Stroke 42: 196–203. 7. 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Thus, the elevated p53 acetylation in sham LTED animals in our current study may provide an explanation for our previous report of enhanced susceptibility of LTED animals to ischemic stress. In conclusion, the current study demonstrates that p53 under- goes enhanced acetylation in the hippocampal CA1 region following GCI, and that the neuroprotective hormone, 17b2E2, acts to strongly suppress the acetylation of p53, leading to ubiquitination and attenuation of p53 levels, as well as attenuation of the p53-regulated pro-apoptotic factors, Puma and Noxa. As a whole, the studies enhance our understanding of p53 regulation in the brain following GCI, and suggest a potentially important regulatory role for estrogen in the control of p53. Supporting Information Figure S1 A) Lack of effect of estrogen on basal Acetyl p53- Lysine373 levels and Puma levels in sham non-ischemic control animals. Estrogen (E2) has no significant effect upon Acetyl p53- Lysine373, p53, and Puma levels in the hippocampal CA1 region of non-ischemic sham control animals as compared to placebo (Pla) treated animals. B) Estrogen attenuates Acetyl p53 (Lysine382) levels at 3 hours following ischemic reperfusion. Acetylation of p53 at Lysine382 did not change in Pla and E2-treated shams. A significant increase in p53 acetylation at Lysine382 is observed in Pla-treated animals at 3 h after ischemic reperfusion, whereas E2- treatment significantly attenuated this elevation. Total p53 levels remained unchanged in all treatment groups examined. *p ,0.05 vs. sham and #p ,0.05 vs. Pla group. (TIF) Author Contributions Conceived and designed the experiments: DWB QZ. Performed the experiments: LR QZ DH YD LD. Analyzed the data: LR QZ DWB. Wrote the paper: LR DWB QZ. 9. Jover-Mengual T, Miyawaki T, Latuszek A, Alborch E, Zukin RS, et al. 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