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. 2013 Sep 17;170(3):489–505. doi: 10.1111/bph.12111

Figure 4.

Figure 4

URB597 and AEA induce Nrf2 nuclear translocation. (A, B) NAC blocks URB597 and AEA from activating nqo1-ARE-Luc reporter. MCF-7 cells co-transfected with 0.5 μg of nqo1-ARE-Luc reporter and 0.15 μg of pCMV-β-galactosidase were pretreated with 1.0 mM of NAC for 1 h, followed by URB597 (5.0 and 10.0 μM) and AEA (2.5 and 5.0 μM) treatment for 16 h. Luciferase activity assay and data analysis were performed as in Figure 1C. The data represent the mean ± SD of at least three independent experiments performed in triplicate. (C, D) NAC inhibits URB597- and AEA-induced HO-1 mRNA expression. MCF-7 cells were pretreated with 5 mM of NAC, followed by 5 μM of URB597 and 2. 5 μM of AEA treatment for 6 h, as indicated. Semi-quantitative RT-PCR for HO-1 mRNA expression was performed as in Figure 1A. (E) URB597 and AEA induce the nuclear translocation of Nrf2 protein in MCF-7 cells and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with URB597 (5 μM) and AEA (2.5 μM) for 6 h, followed by the nuclear fractionation. Furthermore, 10 μg of the nuclear proteins were applied to detect the levels of Nrf2 protein. The same membrane was immunoblotted by anti-lamin B1 antibody to monitor equal loading of proteins. The levels of Nrf2 protein were normalized to lamin B1 levels and the relative expression was represented graphically. All the figures are representative of three independent experiments. *P < 0.05 as compared with the control untreated cells; **P < 0.001 as compared with control untreated cells.