Investigating Nectin4 in Derp1-Induced Bronchial Epithelial Cell Barrier

Primary normal human bronchial epithelial (NHBE) cells (CC-2540, Lonza, Walkersville, MD) (3500cells/cm²) were maintained as previously described (19 (link)). Cells were placed in bronchial epithelial cell growth medium (BEGM, CC-3170, Lonza) without supplements for 24 h and then stimulated with 10μg/ml Dermatophagoides pteronyssinus 1 (Der p1, Arthropods of Medical Importance Resource Bank, Institute of Tropical Medicine, Yonsei University). In separated tests, NHBE were transfected with small interfering RNA (siRNA) duplexes designed against Nectin4 or nonspecific siRNA control (1027418, Qiagen, CA, USA). NHBE cells cultured in 6-well plates were transfected with 100 nM siRNA or negative control using Lipofectamine 2000 (11668019, Invitrogen, CA, USA). After 24hr, cells were treated with 10 μg/ml Der p1 and harvested for western blotting. Trans-epithelial electrical resistance (TEER) was measured using an Epithelial Volt/Ohm Meter (EVOM) (EVOM2, World Precision Instruments, FL). TEER values are expressed by raw oh m values minus the oh m value of a blank insert, multiplied by the area of the insert (1.12 cm²).