HEK293 Cell Culture and PAO Treatment

Human embryonic kidney (HEK293) cells were purchased from the American Type Culture Collection (CRL-1573) and maintained in MEM medium (HyClone, Logan, UT) containing 10% fetal bovine serum (Sigma, St. Louis, MO), 1% antibiotic/antimycotic solution (Mediatech, Manassas, VA) at 37 °C in a 5% CO₂ incubator. PAO (Sigma) stock solution was freshly prepared for each exposure in 100% DMSO as described previously.^{14 (link)} PAO treatment of HEK293 cells was done employing various concentrations (0–250 nM) diluted in the culture medium. Vehicle (alone)-treated cells served as controls. Cobalt protoporphyrin (CoPP) (Enzo Life Sciences, Farmingdale, NY), an inducer of HO-1, was used in some experiments as described in the figure legends. In these experiments, cells were pretreated with CoPP (10 μM) for 6 h before PAO challenge and then both treatments were continued for 24 hours.