Estrogen Receptor and PI3K/Akt Signaling Pathway Involvement in S-(-)Equol-Induced Activation of Nrf2/ARE in Endothelial Cells (original) (raw)
Figure 4
ERβ plays a role in activation of Nrf2 by S-(-)equol in endothelial cells.
(A) EA.hy926 cells were transfected with ARE-dependent firefly luciferase reporter gene (F) and the renilla firefly luciferase gene (R), and treated with 100 nM S-(-)equol in the presence or absence of 10 nM DPN, 10 nM PPT, 100 nM MPP or 100 nM PHTPP for 2 h following further incubation with or without 100 nM S-(-)equol. The potency of induction is expressed as the relative luminescence (R/F) measured using the dual luciferase reporter assay system (mean ± SD, n = 6). *p<0.05 versus with control group, #p<0.05 versus with S-(-)equol treated group. (B) EA.hy926 cells were incubated with 0.1% DMSO (1), 10 nM PPT (3), or 100 nM S-(-)equol (5) for 16 h and then immunostained with an antibody against ERα; and cells also were incubated with 0.1% DMSO (2), 10 nM PPT (4), or 100 nM S-(-)equol (6) for 16 h and then immunostained with an antibody against ERβ, then analyzed by confocal microscopy. (C) Cells were treated with 100 nM S-(-)equol in the presence or absence of 10 nM DPN, 10 nM PPT, 100 nM MPP or 100 nM PHTPP for 2 h following further incubation with or without 100 nM S-(-)equol, and Nrf2 localization was analyzed by confocal microscopy. Values (intensity of nuclear versus cytoplasmic) are means of counting 100 cells with standard deviations represented by vertical bars. *p<0.05 versus with control group, #p<0.05 versus with S-(-)equol treated group.