Fig 3.

Effects of norisoboldine (NOR) on the crosstalk between aryl hydrocarbon receptor (AhR) and NF-κB signaling pathway in osteoclastogenesis. (A) Effects of resveratrol (Res) and α-naphthoflavone (α-NF) on the inhibition of nuclear translocation of NF-κB-p65 through NOR in RANKL-induced RAW264.7 cells. The cells were treated with Res (1 µM), α-NF (1 µM), NOR (30 µM), NOR (30 µM)+Res (1 µM), and NOR (30 µM)+α-NF (1 µM) for 24 h in the presence or absence of RANKL (100 ng/mL), respectively. The cells were subsequently stimulated with RANKL (100 ng/mL) for 15 min. The expression of NF-κB-p65 in the cytosol and nucleus were analyzed using western blotting. (B) Effect of NOR on the accumulation of the AhR-NF-κB-p65 complex in RANKL-induced RAW264.7 cells. The time course of the accumulation of AhR-NF-κB-p65 complex was investigated. RAW264.7 cells were treated with RANKL (100 ng/mL), and the cell lysates were collected at the indicated time points. The cell extracts were immunoprecipitated with an anti-AhR antibody. Co-precipitated NF-κB-p65 was detected through immunoblotting with the relevant antibody. Subsequently, RAW264.7 cells were treated with NOR (10 and 30 μM) for 24 h, and subsequently stimulated with RANKL (100 ng/mL) for 15 min. The cells were lysed, and the extracts were immunoprecipitated with an anti-AhR antibody. Co-precipitated NF-κB-p65 was detected through immunoblotting with the relevant antibody. The data were expressed as the means ± S.E.M. of three independent experiments. ^##p< 0.01 vs. normal; ^*p< 0.05, ^**p< 0.01 vs. control; ^$$p < 0.01 vs. NOR (30 µM).