Nrf2 luciferase reporter assay.

Increase in antioxidant response element (ARE) activity is strongly associated with Nrf2 nuclear translocation. Therefore, to assess whether OD could induce Nrf2 nuclear translocation in epithelial cells, we used human bronchial epithelial cells (BEAS-2B) stably transfected with a pGL4.28 plasmid (Promega, Madison, WI) containing an antioxidant response element (ARE; G TAC CGC AGT CAC AGT GAC TCA GCA GAA TCG CTA G) upstream of a hygromycin B resistance gene and a firefly luciferase construct. Cells were grown in DMEM with 10% FBS, 1% PSA, and 100 µg/ml Hygromycin B and seeded at a density of 70,000 cells/well in 12-well plates for 24 h. Different concentrations (10 or 100 µg/ml) and incubation times (30 min, 2 h, 4 h, 24 h) of OD were used to evaluate whether increases in ARE activity were dose- and time-dependent. In subsequent experiments, cells were pretreated with 5 µg/ml cytochalasin D (Sigma Aldrich) to determine whether inhibition of phagocytosis could prevent changes in Nrf2 nuclear translocation. To assess Nrf2 nuclear translocation after OD treatment, culture medium was removed and cells were gently washed two times with sterile PBS, followed by the addition of 40 µl of reporter lysis buffer (Promega, Madison, WI). Plates were placed on a rocker at 4°C for 5 min followed by collection of whole cell lysates that were spun at 13,000 rpm for 3 min. Supernatant (10 µl) was transferred to a 96-well plate for reading in the Tecan iControl luciferase system in the presence of 470 µM D-Luciferin and 530 µM ATP. Nrf2 nuclear translocation was reported in relative light units.