Non-tumorigenic immortalized human bronchial epithelial HBEC-30 cells ((S1); kindly provided by M. White), were grown in keratinocyte serum free medium supplemented with recombinant epidermal growth factor and bovine pituitary extract (Gibco). All other cell lines were grown in Dulbecco’s Modified High Glucose Eagle Medium (HyClone). Tetracycline inducible cells were supplemented with 10% tetracycline-free fetal bovine serum (Clontech) while all other cells were supplemented with 10% fetal bovine serum (Atlanta Biologicals). Low (1%) O2 incubations were carried out in a hypoxic chamber (Coy Laboratory Products). Transient transfections of plasmid DNA were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Typically, 5×104 cells were plated per well of a 24-well plate and incubated for 16 hr followed by transfection with 500 ng of DNA. Neomycin-resistant cells stably expressing epitope-tagged IRP2 or FBXL5 proteins were isolated over two rounds of clonal selection in the presence of 400 μg/mL G418 (Research Products Inc). IRP1 and IRP13C>3S Flp-In T-Rex HEK 293 cells were kindly provided by R. Eisenstein.
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