Nci h292

All cell lines A-549 (RRID : CVCL_0023), NCI-H1975 (RRID : CVCL_1511), NCI-H292 (RRID : CVCL_0455), HEK293T (RRID : CVCL_0063), NCI-H1993 (RRID : CVCL_1512) were purchase from ATCC and identified by short tandem repeat (STR) profiles from the China Center For Type Culture Collection (CCTCC, Wuhan University, China). All experiments were performed with mycoplasma-free cells. Cells were cultured with Dulbecco’s modified Eagle’s medium media (Thermo Scientific, USA) with 10% fetal bovine serum (FBS, Thermo Scientific, USA). HEK-293T cells were cultured in a six-well plate with complete culture medium the day before and transfected when the cell confluence was approximately 90%. We used a plasmid lentiviral packaging system, and the two packaging plasmids psPAX2 and PMD2.G (Thermo Scientific, USA) and the target gene were mixed at 3 μg:2 μg:1 μg. Then, polyethylenimine (PEI) was added to a quarter of the plasmid. Finally, 200 μL Opti-MEM medium was added to the mixture, and then the mixture was evenly added to the cells. After 6-8 hours, the solution was changed to complete medium. After 48 hours, the cell culture supernatant was collected and filtered with a 0.4-μm filter membrane to obtain virus solution.

The LPS was purchased from Merck. Purified cytokines and specific ELISA kits were purchased from R&D Systems. The PDZ peptides were synthesized by Peptron (Daejeon, Korea). BEAS-2b and NCI-H292 cells were purchased from ATCC. Six- to eight-week-old C57BL/6 mice were maintained in accordance with the guidelines and under the approval of the Animal Care Committee of Kosin University College of Medicine, Busan, Korea.

NCI-H292 (ATCC-CRL-1848; Rockville, MD, USA), BEAS2-B (ATCC-CRL-9609) and A549-NF-κB luciferase cells (Panomics, Fremont, CA, USA) were grown as previously (17 (link), 18 (link)). Primary hAECBs from CF patients or healthy donors (Table 1) were commercially obtained (Epithelix, Plan les Ouates, Switzerland) and were cultured as previously (19 (link)). The cells were seeded in different plates (TPP, Techno Plastic Products, Trasadingen, Switzerland) and after reaching confluence and were incubated overnight in DMEM containing 10% FBS and 1% antibiotics before being stimulated in the same medium free of antibiotics.

Unless otherwise stated, all experiments were performed using human alveolar epithelial cells NCI-H292 (ATCC CRL-1848). Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Wisent, Quebec) at 37°C at 5% CO₂. Cells were cultured and transfected with indicated constructs using FugeneHD for at least 18 h before being used in experiments. MDCK cells (Madin-Darby Canine Kidney) (ATCC CCL-34) were maintained in Dulbecco's modified Eagle's media (DMEM) media supplemented with 10% FBS (Wisent, Quebec) at 37°C at 5% CO₂. 16HBEo14o- cells were originally from Dr. Dieter C. Gruenert (Cardiovascular Research Institute, University of California, San Francisco, USA) (Gruenert et al., 1995 (link)). Cells were grown in Eagle's Minimum Essential Media (EMEM) supplemented with 10% FBS at 37°C at 5% CO₂.