Crl 4051

NSCLC cell lines (A549 (RRID:CVCL_0023), Calu6 (RRID:CVCL_0236), Calu6 with stable hTERT expression (derived in laboratory) and NCI-H1299 (RRID:CVCL_0060)) were maintained in culture at 37°C in 5% CO₂ in 4:1 DMEM:Medium 199 supplemented with 10% cosmic calf serum (HyClone, Logan, UT). All unmodified cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). Human bronchial epithelial (HBEC, primary ATCC - PCS-300–010) and HBEC3-KT cells (ATCC - CRL-4051, RRID:CVCL_X491) were maintained in bronchial epithelial growth media (ATCC - PCS-300–030) supplemented with a bronchial epithelial cell growth kit (ATCC - PCS-300–040) on collagen coated plates (porcine gelatin, Sigma). Cell line identity was verified by the vendor (ATCC). All cell lines were confirmed to be myocoplasma free at the start of the culture and several clean vials were frozen back for subsequent use (e-Myco kit, Bulldog-Bio) and no further testing was completed. Cells were continuously cultured for 70 passages or up to three months’ time, whichever occurred first, at which point a new mycoplasma free vial was obtained, thawed and cultured.

The Human bronchial epithelial cells (HBEC3-kt) cells used in this study are Homo sapiens, female cells with ATCC Cat# CRL-4051, RRID:CVCL_X491. HBECs were grown in Keratinocyte-SFM medium (GIBCO, 17005-042) with 1% penicillin-streptomycin (ThermoFisher, 15140122). For live cell imaging, we used Airway Epithelia Cell Basal Medium (ATCC, PCS-300-030) supplemented with Bronchial Epithelial Cell Growth Kit (ATCC, PCS-300-040) to minimize the phenol-red interference. The polyclonal and single-cell clone cell lines with MS2 stem-loop intronic labeling were derived from this HBEC3-kt parental cell line. They were cultured in the same conditions.