Pcs 301 010

Primary normal human lung fibroblasts (NHLFs) were purchased from Lonza and were maintained in manufacturer supplied growth medium (FGM-2 BulletKit, CC3132, Lonza). NHLFs were cultured up to six passages with media change every 3 days. Human primary SAEC (PCS-301-010, ATCC) were maintained in manufacturer supplied growth medium (PCS-300-030 and PCS-300-040, ATCC) supplemented with 100 U/ml penicillin, and 100 µg/ml streptomycin.

NSCLC cell lines were obtained from the US National Cancer Institute (Bethesda, MD, USA) (MTA 1-2702-09). All cells were incubated at 37 °C and maintained at 5% CO₂. H23, H226, IMR90 (normal lung fibroblast, ATCC CCL-186) and Lung Primary (Primary Small Airway Epithelial Cells; Normal, Human, ATCC PCS-301-010) cell lines were obtained from ATCC. IMR-90 cell was grown in DMEM/HIGH GLUCOSE medium (SH30243.01, Hyclone, Logan, UT, USA) containing 10% FBS. Lung primary cell was airway epithelial cell basal medium (PCS-300-030, ATCC, Manassas, VA, USA) with the bronchial epithelial cell growth kit (PCS-300-040, ATCC, Manassas, VA, USA).
NSCLC cells were grown in RPMI 1640 medium (Hyclone, Logan, UT, USA) plus 10% fetal bovine serum (FBS; Hyclone), penicillin and streptomycin. A small interfering RNA (siRNA) duplex targeting human GLS1, GOT2 and MDH2 (Santa Cruz, CA, USA) was introduced into the cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. As negative controls, cells were incubated with Lipofectamine 3000 (Invitrogen) and a negative siRNA (Santa Cruz). The hypoxic condition was achieved by incubating the cells in 1% of O₂, 94% of N₂ and 5% of CO₂ in a multigas incubator (Vision scientific. VS-9000GC, Seoul, Korea).

Human primary small airway epithelial cells (SAEC, PCS-301-010™) were purchased from ATCC^® (Manassas, VA, USA) and cultured using the air–liquid interface (ALI) cell culture system. The media and reagents for the ALI system, including PneumaCult™-Ex Plus Medium (#05002), PneumaCult™-ALI Basal Medium (#05002), PneumaCult™-ALI 10X Supplement (#05003), and PneumaCult™-ALI Maintenance Supplement 100X (#05006) were purchased from STEMCELL Technologies (Vancouver, BC, Canada). The CelTox Sampler was purchased from MedTec Biolab Inc. (Hillsborough, NC, USA) for the in vitro exposure assessment. For the toxicological assays, CellTiter 96 Aqueous One Solution (Promega, Madison, WI, USA) and GSH-Glo^TM Glutathione reagent (Promega, Madison, WI, USA) were used to measure cellular viability and total glutathione, respectively, in SAEC.

Primary small airway epithelial cells (SAEC) (ATCC^® PCS-301-010™, Manassas, VA, USA) were cultured in the T-75 cell culture flask using PneumaCult™-Ex Plus Medium (STEMCELL, Vancouver, BC, Canada) and incubated at 37 °C until 70–80% confluency. For the pod-type ENDS (JUUL^®) aerosol exposure, SAEC was subcultured into the trans-well inserts within a 24-trans-well cell culture plate at 10,000 cells/mL density. The subcultured cells were maintained by changing the media every 2–3 days for approximately 14–21 days until a monolayer was formed. Once the monolayer was formed, the cells were air-lifted by removing the media from the apical surface. During the air-lifted phase, the cells were maintained by changing the basolateral media (PneumaCult™-ALI media, STEMCELL, Vancouver, BC, Canada) every 2–3 days. Each exposure (blank, puffs 1–50, puffs 101–150) was administered directly onto the apical surface (100 µL) and incubated at 37 °C for 24 h and 7 days. During the 7-day exposure, all the exposures were changed every day, and the basolateral media (PneumaCult™-ALI media, STEMCELL, Vancouver, BC, Canada) was changed every 2–3 days. In this study, all cytotoxicity assays employed negative (untreated) and positive controls. For positive control, 0.1% hydrogen peroxide was administered onto the apical surface (100 µL) and incubated at 37 °C for 20 min prior to each assay.