To confirm the Nrf2 activation potential, ARE promoter Responsive Luciferase Reporters such as GST-ARE-Luc and hNQO1-ARE-Luc reporter gene constructs were used in endothelial cells. Briefly, EA.hy926 cells were seeded and transfected with an ARE-Luc construct (500 ng/well) with the help of Lipofectamine 2000. After 24 h, the transfected cells were administered with different amounts of RA (0-20 μM) and kept incubated for 8 h, and then the cells were assayed for luciferase activity by a luminometer (Promega, Madison, WI, USA). The luciferase activity was expressed in terms of relative fold variation compared to control cells.

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