Gnf351

The SH-SY5Y cell line was kindly provided by Dr. Cinzia Mallozzi (ISS, Rome, Italy) [42 (link)] and maintained in culture in Dulbecco’s modified Eagle medium (DMEM)/nutrient mixture F-12 (Merk/Sigma-Aldrich, Italy) supplemented with 10% FBS (GIBCO Life Technologies, Grand Island, NY, USA), 1% Glutamine and 1% Penicillin–Streptomycin (Merck/Sigma-Aldrich, Italy) at 37° C in a humidified incubator with 5% CO₂. To study AHR nuclear translocation, cells were plated in 100 mm diameter dishes (1 × 10⁶ cells), maintained in culture conditions for 48 h, and stimulated for different time lengths (15 min (min), 30 min, 2 h, 6 h) with 100 µM of EDA (Merk/Sigma-Aldrich, Italy) or 1 µM INDI (Merck/Sigma-Aldrich, Italy). For CYP1a1 and NRF2 protein expression analysis, cells were treated for 24 h with EDA 100 µM or INDI 1 µM. To inhibit the AHR nuclear translocation, cells were treated with 1 µM of the AHR antagonist III GNF351 (Merck/Sigma-Aldrich, Italy) for 15 min before the addition of EDA or vehicle alone (DMSO).

VRCZ and VNO were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). They were dissolved in DMSO as 150 mM stock solutions and were used immediately or stored at −20 °C until used. HPLC-grade acetonitrile and methanol were purchased from Wako Pure Chemicals (Osaka, Japan). Photoproduct of VNO (P-VNO) was generated from VNO by irradiating with UVB irradiation (500 mJ/cm²). Afloqualone (AQ) was obtained from Tanabe Seiyaku Co., Osaka, Japan. The solution of PBS contained 0.3 mM AQ was prepared as described previously^{20 (link)}. Sparfloxacin, the AhR antagonists CH223191 and GNF351 were procured from Sigma-Aldrich, St. Louis, MO. KCZ, TBF and ITCZ were purchased from Wako Pure Chemicals, Osaka, Japan. Recombinant human TNF-α was purchased from R&D Systems, Minneapolis, MN.