Figure 3.

YD1 activates Nrf2-ARE signaling. RAW 264.7 cells were treated with YD1 (10 μg/mL) for the indicated time durations, and ARE luciferase activity was measured by a Dual Luciferase reporter assay as described in Materials and Methods (A). The levels of Nrf2 protein in cytosolic and nuclear fractions were analyzed by Western blot analysis (B). RAW 264.7 cells were treated with YD1 (10 μg/mL), and protein levels of HO-1 (upper panel) and NQO1 (lower panel) were analyzed by Western blotting (C). Densitometric analysis was carried out to quantify the band intensity by β-actin normalization. Data (mean ± SD) were representative of at least three independent experiments and expressed as the fold-induction relative to untreated cells (at time zero); ** p < 0.05 versus untreated control. RAW 264.7 cells were transiently transfected with Nrf2 siRNA for 24 h followed by YD1 treatment and incubated for 6 h. Nrf2 protein levels were measured by Western blotting analysis (D). Cells were pretreated by YD1 (2.5, 5, and 10 μg/mL) for 12 h, and subsequently stimulated by LPS for 24 h. The production of intracellular ROS was assayed using the fluorescent probe DCFH-DA (E). Immunofluorescent confocal microscopy showed the level of ROS (green fluorescence) in RAW 264.7 cells (F). Cells were treated with brusatol (a pharmacologically specific Nrf2 inhibitor) for 30 min, followed by YD1 and (10 μg/mL) and gallic acid (GA) (10 μg/mL) for 12 h and then subjected to LPS insult for 24 h, and the cellular ROS generation was measured (G). ^# p < 0.05 compared to the untreated cells; ** p < 0.05 compared to UVB-treated cells. ^$ p < 0.05 compared with YD1 and gallic acid-treated cells.