Fig 2.

Activation of aryl hydrocarbon receptor (AhR) through norisoboldine (NOR). (A) Molecular docking of NOR in the homology model of the AhR ligand binding domain. (B) Effect of NOR on the nuclear translocation of AhR. RAW264.7 cells were treated with NOR (3, 10 and 30 μM) for 24 h and subsequently lysed. The expression of AhR in the cytosol and nucleus was analyzed using western blotting assay. (C) Effect of NOR on the accumulation of the AhR-ARNT complex in RAW264.7 cells. RAW264.7 cells were treated with NOR (3, 10 and 30 μM) for 24 h and subsequently lysed. The cell extracts were immunoprecipitated with anti-AhR antibody. Co-precipitated ARNT was detected through immunoblotting. (D) Effect of NOR on AhR-mediated reporter gene activity. RAW264.7 cells were transiently transfected with a luciferase reporter gene expression vector under control of a promoter containing xenobiotic response elements (XRE-Luc) and treated with NOR (3, 10 and 30 μM) for 24 h. The absorbance at 405 nm was read using a Spectromax 96-well plate reader. (E) Effect of NOR on the mRNA expression of CYP1A1 in RAW264.7 cells. The cells were treated with NOR (3, 10 and 30 μM), and the cell lysates were collected at the indicated time points. The mRNA expression of CYP1A1 was detected using Q-PCR. The data were expressed as the means ± S.E.M. of three independent experiments. ^*p< 0.05, ^**p< 0.01 vs. normal.