Supplementary growth factors

HOK-16B cells (RRID: CVCL_B405) were maintained in keratinocyte growth medium (KGM) containing supplementary growth factors (Lonza, Basel, Switzerland) at 37 °C in a water-saturated atmosphere of 5% CO₂. To examine the effect of the clinical isolates on the cell viability and proliferation of HOK-16B cells, cells (6 × 10⁴ cells) were infected with four clinical isolates of E. coli, E. coli BL21 (Thermo Fisher Scientific, Waltham, MA, USA), and S. salivarius KCTC 5512 (Korean Collection for Type Culture, Jeongeup, Jeollabuk-do, Korea) at a multiplicity of infection (MOI) of 1000 in KGM without antibiotics for 2 hours. After 2 hours, 50 μg/ml and 100 μg/ml gentamicin were added to kill extracellular E. coli and S. salivarius, respectively. After incubation for the indicated times, the cells were detached by trypsin-EDTA and enumerated after trypan blue staining. To analyze apoptosis of cells, the detached cells were also washed with Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, pH 7.4) and stained with 5 μl of Annexin V-Alexa Fluor™ 488 conjugate (Life Technologies, Carlsbad, CA, USA) and 5 μg/ml PI in 100 μl of Annexin-binding buffer for 15 min at room temperature. After adding 400 μl of Annexin-binding buffer, the stained cells were analyzed by FACSCalibur™ (BD Biosciences, San Diego, CA, USA).

RPMI 1640 medium was purchased from Biochrom (Berlin, Germany). FCS was from Life Technologies (Carlsbad, CA, USA). Penicillin and streptomycin were from PAA Laboratories (Pasching, Austria). PBS was obtained from PAN-Biotech (Aidenbach, Germany). EBSS was from Sigma-Aldrich (Saint Louis, MO, USA). Bronchial epithelial basal medium (BEBM) and supplementary growth factors were all from Lonza (Walkersville, MD, USA). PureCol (type I bovine collagen solution, 3 mg/ml) was from Advanced BioMatrix (San Diego, CA, USA). Soybean trypsin inhibitor (STI), DNase I, and Protease XIV were from Sigma-Aldrich. Primary antibodies detecting p-STAT1 and actin were all from Cell Signaling Biotechnology (Frankfurt, Germany). IFNλ antibody was from abcam (ab38569, Cambridge, UK).