Sulforaphane sf

HPI-4, bixin, and sodium arsenite (As(III)) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and sulforaphane (SF) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Recombinant Human Shh was purchased from PeproTech (Rocky Hill, NJ, USA). MEFs were isolated from Nrf2 WT (Nrf2^+/+) and knockout (Nrf2^−/−) mice and cultured with DMEM (Corning, Corning, NY, USA) supplemented with 10% FBS (Atlanta Biologicals, Bio-Techne, Minneapolis, MN, USA), 1% L-glutamine (Invitrogen, Carlsbad, CA, USA), 1% Nonessential Amino Acids (Invitrogen), 0.1% beta-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Invitrogen). H838 and H1299 were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. BEAS-2B cells were cultured in Ham’s F-12 medium supplemented with 1% bovine hypothalamus extract (PromoCell, Heidelberg, Germany), insulin (2 mg/ml; Sigma-Aldrich), epidermal growth factor (10 μg/ml; Millipore, Burlington, MA, USA), transferrin (2.5 mg/ml; Sigma-Aldrich), cholera toxin (10 μg/ml; List Biological Laboratories, Inc., Campbell, CA, USA), and dexamethasone (0.05 mM; Sigma-Aldrich). For primary cilia analysis, cells were cultured with DMEM containing 0.5% FBS and 1% penicillin/streptomycin for 48 h. All cells were cultured at 37°C in a humidified incubator containing 5% CO2.

Bixin, tBHQ, and sodium arsenite (As(III)) were purchased from Sigma, and sulforaphane (SF) was from Santa Cruz. Primary antibodies against NRF2, KEAP1, GCLM, HO-1, GAPDH, and the hemagglutinin (HA) epitope, as well as horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz. Antibodies against p-P65 and P65 were from Cell Signaling, and the 8-oxo-dG antibody was from Trevigen. The Alexa Fluor 488-conjugated secondary antibody was from Invitrogen. The human non-small cell carcinoma H1299 cells were purchased from ATCC and were grown in RPMI 1640 medium supplemented with 10% FBS (Atlanta Biological) and 0.1% gentamycin (Invitrogen). Normal human bronchial epithelial cells (NHBE) and normal lung microvascular endothelial cells (HMVEC-L) were purchased from Lonza and were grown in Bronchial Epithelial Growth Medium (BEGM, Lonza) and Endothelial Cell Growth Medium (EGM, Lonza), respectively, according to the supplier’s instructions. All cells were maintained at 37 °C in a humidified incubator containing 5% CO₂.