Permeable membrane supports

Passage 1 primary hBECs were prepared in permeable membrane supports (12-well; 0.4 µm pore size, Corning Incorporated, Corning, NY) as previously described [8] (link), [9] (link). After 24 hrs, BEGM medium was removed from both the upper and lower chamber, and medium in the lower chamber only was replaced with 1 mL of air-liquid interface (ALI) medium, consisting of a 50∶50 mixture of BEGM and DMEM (Mediatech, Manassas, VA) that had been supplemented with all the BEGM additives (Lonza) except retinoic acid. To this mixture, fresh all-trans retinoic acid (50 nM, Sigma-Aldrich) was added, along with either 1,25(OH)₂D or 25(OH)D (0.1, 1, 10, and 100 nM, Sigma-Aldrich). Since serum levels of 30–80 ng/mL (75–200 nM) 25(OH)D are considered optimal for health, with levels of 1,25(OH)₂D being dependent upon the degree of localized synthesis, the range of concentrations used in our experiments did not exceed the physiologic range [1] (link). Medium with retinoic acid was replaced daily during the first week, and then every 2–3 days. Transepithelial resistance measurements were made using a voltohmmeter.

Airway epithelial cells (H441) were grown on permeable membrane supports (Transwells, Corning, NY, USA) until confluent and then taken to air‐liquid interface to form polarized monolayers, as previously described.21 Monolayers were pretreated with 1 mM metformin (pharmacological dose to elicit changes in vitro) which was added to the basolateral medium for 18 hours before apical addition of S. aureus. A single colony of S. aureus strain 8325‐4 was selected and grown overnight in 37°C in RPMI media (Life Technologies, Paisley, UK) and diluted with glucose‐free RPMI to produce a culture of approximately 5 × 10⁵ CFU in 50 μL this was applied to the apical surface of H441 monolayer as described previously.13 Co‐cultures were placed in a CO₂ incubator at 37°C for 7 hours, after which monolayers were immunostained for TJ or cell lysates were prepared for Western blot analysis. Transepithelial electrical resistance (TEER) was measured using a voltohmmeter (WPI, Hitchin, UK). H441 monolayers were pretreated with protein kinase inhibitors (50 nM staurosporine or 10 μM PKC pseudosubstrate or 80 μM Compound C were prepared in dimethyl sulfoxide) 30 minutes prior to the addition of metformin.