Estrogen Receptor and PI3K/Akt Signaling Pathway Involvement in S-(-)Equol-Induced Activation of Nrf2/ARE in Endothelial Cells (original) (raw)
Figure 3
The effects of PI3K/Akt and ER inhibitors on S-(-)equol-induced Nrf2 activation in endothelial cells.
(A) EA.hy926 cells or HUVECs were transfected with ARE-dependent firefly luciferase reporter gene (F) and the renilla firefly luciferase gene (R), and treated with 250 nM S-(-)equol in the presence or absence of 10 µM LY294002 or 100 nM ICI182,780 for 16 h. The potency of induction is expressed as the relative luminescence (R/F) measured using the dual luciferase reporter assay system (mean ± SD, n = 3). *p<0.05 versus with control group, #p<0.05 versus with S-(-)equol treated group. (B) EA.hy926 cells or HUVECs were incubated with or without 10 µM LY294002 or 100 nM ICI182,780 for 30 min and then with or without 250 nM S-(-)equol or 500 nM daidzein for 24 h. Whole cell lysates were then immunoblotted with antibodies against Nrf2, HO-1, NQO1, and β-tubulin. Values are means of three independent experiments with standard deviations represented by vertical bars. Mean values were significantly different compared with controls (*p<0.05). (C) EA.hy926 cells or HUVECs cotransfected with the HA-Nrf2 and renilla firefly luciferase expression plasmids were treated with 250 nM S-(-)equol in the presence or absence of 10 µM LY294002 or 100 nM ICI182,780 for 16 h, and HA-Nrf2 localization was analyzed by confocal microscopy. (D) EA.hy926 cells were incubated with 250 nM S-(-)equol, 500 nM daidzein with or without 10 µM LY294002 or 100 nM ICI182,780 for 16 h and then immunostained with an antibody against Nrf2, 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.