Primary antibodies against nrf2

PD (purity above 98%) was supplied by Guangzhou Honsea Sunshine Biotech Co. Ethanol and silymarin were purchased from Sigma-Aldrich (purity above 98%, St Louis, Missouri, USA). Ethanol was diluted with distilled water to 56% (v/v). PD and silymarin were suspended in 0.5% carboxymethylcellulose (CMC-Na) distilled water solution. Triglyceride (TG) was purchased from Beijing BHKT Clinical Reagent Co. Ltd (Beijing, China). Primary antibodies against Nrf2, HO-1, CYP2E1, TLR4, NF-κB p65, and β-actin and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other required materials are obtained from Sigma Co. LLC. (Guangzhou, China) with highest purity commercially available.

EP, LP, undifferentiated, differentiated, OTA-, or t-BHQ-treated MSCs were seeded at 5000 cells/cm² on 4-well glass chamber slides (Nalge Nunc International, Rochester, NY, USA), and the cells were incubated in a 5% CO₂ incubator at 37 °C. After an overnight incubation, the cells were washed with PBS followed by fixation with 4% paraformaldehyde (Sigma) for 30 min. Permeabilization was accomplished with 1% Triton X-100 in PBS for 10 min followed by blocking for 1 h with 3% bovine serum albumin (BSA) in PBS. The cells were incubated with a 1 : 200 dilution of primary antibodies against NRF2 (Santa Cruz Biotechnology) overnight at 4 °C. After washing three times with PBS, the cells were incubated with fluorescein isothiocyanate (FITC) and phycoerythrin-conjugated secondary antibodies (Santa Cruz Biotechnology) or Alexa Fluor 568 (Yellow, Abcam) in a 1 : 5000 dilution in 3% BSA-containing PBS for 1 h at room temperature in the dark. The nuclei were stained with 4,6-diamidino-2-phenyindole (DAPI, Sigma) and then examined using a Zeiss LSM700 scanning laser confocal microscope (Zen 2011; Carl Zeiss MicroImaging GHBH, Jena, Germany).

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.