(A) ONHAs were pretreated with 25 μM estrogen followed by induction of oxidative stress using tBHP. Production of NFTs was determined by staining with Thioflavin S (green). (B) tBHP treated cells showed significant Thioflavin S staining, indicative of NFT formation, compared to mock (*, p=0.0129). Cells pretreated with estrogen for 2 h showed a significant reduction of Thioflavin S staining compared to cells treated with tBHP treated (*, p=0.0171). In addition, NFTs appeared hallo like and more diffuse for the 2h pretreatment. Increasing pretreatment time to 18 h with estrogen reduced NFT formation significantly (*, p=0.0134) compared to tBHP treated cells. Pretreatment with estrogen for 18 h was significantly more effective than the 2 h pretreatment (**, p=0.0024). ONHAs pretreated with estrogen for 2 h still had significant NFT formation compared to mock treated (**, p=0.0024). Estrogen pretreatment for 18 h was the most effective showing no significant difference in the level of NFT formation compared with mock treated ONHAs (ns, p=0.1326). Experiments were done in triplicate (n=3) and values were depicted as mean +/− SEM and analyzed using ANOVA (p=0.0007). For statistical comparison, the Bonferroni post-hoc test and Student t-test were used. Statistical significance was set at p ≤ 0.05.