Hyd-activated antioxidant gene transcription via Nrf2/ARE in general; Engineered luciferase promoter containing 3x ARE sites suggested that Hyd activated the Nrf2-dependent transcription through ARE sequences in LECs. (A,B) SRA-hLECs (A) and mLECs (B) were transiently transfected with pRBGP2-3xARE-LUC plasmid or its mutant at all three ARE sites. Then, 14 h later, the transfected LECs were treated with different concentrations of Hyd for 24 h, as indicated. Relative LUC activity was observed. The data represent the mean ± S.D. from three independent experiments. ** p < 0.001. (C) Progressive decline in Nrf2/ARE-mediated transcription with advancing age was reactivated by Hyd treatment. Primary hLECs of different age groups were transiently transfected with pRBGP2-3xARE-LUC plasmid. At 14 h of post-transfection, hLECs were treated with Hyd (20 µM) for 24 h as indicated, and relative LUC activity was quantified. The data represent the mean ± S.D. values obtained from three independent experiments. ** p < 0.001.