*2.2. Treatments*

Aβ (25–35) (Sigma Aldrich, St. Louis, MO, USA) was dissolved in sterile distilled water at a concentration of 1 mM, then incubated in a capped vial at 37 ◦C for 5 days to allow formation of the aggregated form. It was then stored frozen at −20 ◦C until use. 17β-Estradiol, S-equol, and ICI-182,780 (all from Cayman Chemical, Ann Arbor, MI, USA) were dissolved in 99.5% ethanol to make stock solutions, which were used for experiments at a final concentration of 10 nM for estradiol and 1 μM for equol and ICI-182,780 in culture medium. It should be noted that no cytotoxic effect of the vehicle (99.5% ethanol) *per se* on cells was observed via the analysis of cell viability in our preliminary experiments that were conducted to determine the appropriate concentrations of the aforementioned treatments for the present study.

To induce cell death, cells were incubated with (Aβ) or without (C) 1 μM Aβ (25–35) for 24 h. To study the effects of estradiol (E2) and equol (Eq), cells were preincubated with estradiol (E2 + Aβ) or equol (Eq + Aβ) for 24 h prior to Aβ (25–35) exposure. Estradiol was used as a positive control and ICI-182,780 was used as an ER antagonist. It was added 1 h before the estradiol or equol treatment.
