Mice were sensitized on days 1 and 14 by i.p. injection of 20 μg OVA (Sigma-Aldrich, ABT-263 cost St. Louis, MO, USA) emulsified in 1 mg of aluminum hydroxide (Pierce Chemical, Rockford, IL, USA) in a total volume of 200 μL, as previously described with some modifications 9, 48. On days 21, 22, and 23 after

the initial sensitization, the mice were challenged for 30 min with an aerosol of 3% (weight/volume) OVA in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12, Omron, Japan). OVA-treated mice are defined throughout the manuscript as OVA-sensitized and OVA-challenged mice. BAL was performed 48 h after the last challenge as described previously 9. Total cell numbers were counted with a hemocytometer. Smears of BAL cells were prepared with a cytospin (Thermo Electron, Waltham, MA, USA). The smears were stained with Diff-Quik solution (Dade Diagnostics of P. R., Aguada, Puerto Rico) in order to examine the cell differentials. Murine tracheal epithelial cells were isolated under sterile conditions as described Cilomilast previously 48. The epithelial cells were seeded onto 35-mm collagen-coated dishes for submerged culture. The growth medium, DMEM (Invitrogen Life Technologies, Carlsbad, CA, USA), containing 10% fetal bovine serum, penicillin, streptomycin, and amphotericin B was supplemented with insulin,

transferrin, hydrocortisone, phosphoethanolamine, Buspirone HCl cholera toxin, ethanolamine, bovine pituitary extract, and bovine serum albumin. However, DMEM without antibiotics was used as the growth medium for the transfections of siRNA. The cells were maintained in a humidified 5% CO2 incubator at 37°C until they adhered. RNA interference was performed with Stealth RNA interference

(Invitrogen Life Technologies). We transfected primary cultured tracheal epithelial cells in third passage with siRNAs in six-well plates, but not coated with collagen. Stealth siRNA targeting HIF-1α or negative control siRNA was transfected to the cells grown until 30–50% confluence. After the transfections, the cells were incubated for 72 h and then harvested. For transfections, siRNA duplexes were incubated with Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instruction. The sequences of Stealth siRNA were as follows: mouse HIF-1α, 5′-AAGCAUUUCUCUCAUUUCCUCAUGG-3′ (sense); corresponding negative control, 5′-AAGACCUUUAUCUCUUACUCCUUGG-3′ (sense); mouse HIF-2α, 5′-GUCACCAGAACUUGUGCAC-3′ (sense); corresponding negative control, 5′-UAGCGACUAAACACAUCAA-3′ (sense). Cells were seeded in culture dishes and grown until 70% confluence. The medium was then replaced with a new medium containing vehicle (0.1% DMSO), 2ME2 (50 or 100 μmol/L, Calbiochem-Novobiochem, San Diego, CA, USA) for 24 h at 37°C, or IC87114 (2 or 10 μmol/L) for 2 h at 37°C, respectively 40.