Begm growth medium

NHLF were resuspended in FGM-2 growth medium (Lonza), mixed with an equal volume of NHBE in BEGM growth medium (Lonza), and seeded into a 96-well, flat-bottom culture plate (Corning) at a density of 20,000 and 1,600 cells per well, respectively. Compound dilution series were added, and the coculture was incubated for 18 hours at 37°C/5 % CO₂. The medium was then replaced with Fibroblast Growth Basal Medium (Lonza) containing 0.1 % fatty acid–free BSA and the compounds at the indicated concentrations. The medium was supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, for the entire duration of the culture. If not indicated otherwise, the coculture was incubated at 37°C/5 % CO₂ for 96 hours.

Primary nasal epithelial cells were obtained by digesting nasal turbinates or polyps with 0.5 mg/mL collagenase IV (Worthington Biochemical Corp., USA) for 1 hour in Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, NL) followed by an incubation with anti-EpCAM MicroBeads (Miltenyi Biotec, DE) and a positive selection on a magnetic column. Epithelial cells were grown in 75 mL flasks in BEGM growth medium (Lonza Clonetics, NL). Culture medium was replaced every other day. Cells were grown in fully humidified air containing 5% CO₂ at 37°C.
The human airway epithelial cell line NCI-H292 (ATCC, USA) was cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (HyClone, USA), 1.25 mM of glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Creation of the EGR-1 and DUSP-1 knock-down mutants is described elsewhere [18 (link)].

ESCC cell lines including KYSE150, TE-1 (National Collection of Authenticated Cell Cultures, ShangHai, China), KYSE510, TE-7 (Guangzhou Jennio Biotech Co. Ltd., Guangzhou, China), and EC109 (National Infrastructure of Cell Line Resource, Beijing, China). Immortalized human esophageal squamous epithelial cell line Het-1A was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA; Cat# CRL-2692^TM). 293T cell line was purchased from (National Collection of Authenticated Cell Cultures). ESCC and 293T cell lines were cultured in RPMI-1640 and DMEM medium (HyClone™, USA), respectively, supplemented with 10% fetal bovine serum (FBS; Gibco^TM, Thermo Scientific™, USA; Cat# 10099141C), 100 μg/ml streptomycin, and 100 units/ml penicillin (HyClone™). Het-1A cells were grown in BEGM™ Growth Medium (Lonza Walkersville Inc., USA). All cell lines were authenticated by STR and tested for mycoplasma recently.