Figure 3.

Daidzein, daidzein-4′-sulfate and equol effect on ERα activation status. (a) Western blot (top) and densitometric analyses (bottom) of NGB protein levels in MCF-7 cells treated for 24 h with either vehicle (DMSO) or E2 (10 nM) or D, D4S and Eq (1 μM) in presence or absence of the ERα inhibitor Endoxifen (1 μM; 30 min pretreatment). The amount of proteins was normalized by comparison with tubulin levels. Data are the mean ± SD of three different experiments. p < 0.001 was determined with Student’s t test with respect to the vehicle (*) or E2-treated (°) samples. (b) ERα activation by daidzein, daidzein-4′-sulfate and equol. The panel represents the ERαSer118 phosphorylation status calculated as the ratio pERα/ERα). Determined by Western blot analysis in MCF-7 cells exposed for 1h to either vehicle (DMSO) or E2 (10 nM) or D, D4S and Eq (1 μM) in presence or absence of ERα inhibitor Endoxifen (1 μM; 30 min pretreatment). The nitrocellulose was stripped and then probed with anti-ERα antibody. The pERα/ERα ratio was calculated with respect to tubulin obtained by densitometric analyses of three different experiments (mean ± SD). p < 0.001 was determined by Student t test with respect to vehicle (*), E2-treated (°) or Endox-untreated samples (#). DMSO: dimethyl sulfoxide; E2: estradiol; Endox: endoxifen; ERα: estrogen receptor α; NGB: neuroglobin; D: daidzein; D4S: daidzein-4′-sulfate; Eq: equol.