Fig. 4.

The protective effects of CA, QU and AV on UVA-suppressed Nrf2 nuclear translocation and Nrf2-ARE transcriptional activity. (A) Time-dependent effects of UVA (8 J/cm²) on Nrf2 nuclear translocation in B16F10 cells harvested at various times after UVA exposure. Western blot was performed to determine Nrf2 nuclear translocation at 1, 3, 6 and 12 h post-irradiation. Nrf2 was detected at 68 kDa, TATA binding protein (TBP), the loading control for nuclear protein, at 37 kDa and and α-Tubulin, the loading control for cytosol protein, at 50 kDa. Cells treated with 10 µM of sulforaphane (SF) for 6 h was used as positive control. (B) Nrf2 mRNA was assessed at 15 min, 2, 4 and 8 h after UVA exposure. *P<0.05; **P<0.01; ***P<0.001 versus unirradiated control cells. (C) Nrf2 nuclear accumulation was detected in B16F10 cells pretreated with 1 µM of ERK inhibitor (U0126), JNK inhibitor (SP600125) and p38 inhibitor (SB203580) for 1 h before UV exposure and harvested at 1 h post-irradiation. Protection against UVA-dependent reduced (D) nuclear translocation of Nrf2 and (E) Nrf2-ARE activity by test compounds was examined in B16F10 cells at 1 h after UVA irradiation. Nrf2 activity was determined by a dual luciferase assay after transfection of the cells with ARE luciferase reporter construct. The data are represented as means±SD of three independent experiments. The statistical significance of differences between the control and irradiated cells was evaluated by Student’s t test and between UVA-irradiated and compounds-treated cells by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ^###P<0.001 versus unirradiated control cells. *P<0.05; **P<0.01; *** P< 0.001 versus untreated cells exposed to UVA.