Triiodothyronine

PC-3, a CRPC cell line, DU-145, a prostate adenocarcinoma cell line, and PNT2, an immortalized prostate epithelial cell line, were maintained in RPMI1640 medium with 5% to 10% FBS as described previously [4 (link),60 (link),72 (link),73 (link)]. RWPE1, a human normal prostate cell line, was maintained in keratinocyte serum-free medium (ThermoFisher, Waltham, MA, USA) supplemented with recombinant human epidermal growth factor (hEGF) and bovine pituitary extract. Human prostate epithelial cells (PrEC) were maintained in PrEBM basal medium supplemented with bovine pituitary extract, triiodothyronine, insulin, hEGF, hydrocortisone, transferrin, epinephrine, gentamicin sulfate-amphotericin, and retinoic acid (Lonza, Basel, Switzerland).

Immortalized human airway epithelial cells (16HBE14o-) were purchased from Dr. D.C. Greunert (California Pacific Medical Center Research Institute, San Francisco, CA).16HBE14o- (HBE) cells were grown in Bronchial Epithelial Cell Basal Medium (Clonetics™ BEGM™ BulletKit™ (CC-3170) supplemented with: BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml; Transferrin, 0.5 ml; Insulin, 0.5 ml; Retinoic Acid, 0.5 ml; Triiodothyronine, 0.5 ml (Lonza, Walkersville, MD). Cells were cultured in 6 well plates and 2 × 10⁶ cells per well were infected at a MOI of 10 with either fully virulent strain of Y. pestis, CO92, or a derivative avirulent Y. pestis strain CO92 (Pgm-, Pst-), or were treated with heat-killed Y. pestis CO92. Untreated, and E. coli lipopolysaccharide (LPS)-treated cells (100 ng/ml), were also included as controls. Cells were harvested at 30 min, 1, 4, and 8 h post infection, washed with 1 × PBS and then lysed using lysis buffer: 30 ml 2× Novex Tris-Glycine SDS Sample Loading Buffer (Invitrogen), 20 ml T-PER Tissue Protein Extraction Reagent (Thermo Scientific), 200 μl 0.5 M EDTA pH 8.0, 1X Complete Protease Inhibitor Cocktail (Roche), 80 μl 0.1 M Na₃VO₄, 400 μl 0.1 M NaF, 1.3 ml 1 M DTT. Samples were then stored at −20°C.