Pcs 300 040

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).

All NSCLC cell lines, were obtained from the U.S. National Cancer Institute (NCI; MTA no. 2702–09). Cells were incubated at 37°C and maintained at 5% CO₂. H23, H226, 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, Masassas, VA, USA). NSCLC cells were grown in RPMI 1640 medium (SH30027.01, HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (SH30070.03HI, HyClone, Logan, UT, USA), penicillin, and streptomycin. siRNA duplexes targeting human ALDH1L1 (sc-78373), DHFR (sc-37078), GOT2 (sc-60052), and MDH2 (sc-89622) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were introduced into cells using Lipofectamine^® 3000 (L3000015, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. As negative controls, cells were incubated with Lipofectamine^® 3000 alone and a negative siRNA (sc-37007, sc-44230) (Santa Cruz). For ALDH overexpression, p3x FLAG-CMV-ALDH isoform constructs individually expressing ALDH1L1 and DHFR were produced by Cosmogenetech (Seoul, KOREA). Each cDNA sequence of ALDH1L1 and DHFR was obtained from NCBI. The plasmids were transfected into cells using Lipofectamine^® 3000 according to the manufacturer's instructions.

All cell lines were purchased from the American Type Culture Collection (ATCC); A549 (CAT NO. CCL-185), SW480 (CCL-228), lnCAP (CRL-1740), and PC3 (CRL-1435) cell lines were maintained using RPMI-1640 medium (Sigma-Aldrich, R8758). NCI-H460 (HTB-177), HT29 (HTB-38), MCF7 (HTB-22), and MDA-MB-231 (HTB-130) cell lines were kept in high glucose DMEM (Sigma-Aldrich, D5796). Media were supplied with 10% FBS (Biological Industries, 04-007-1A), 100 units/ml of penicillin G and 100 μg/ml of streptomycin (Biological Industries, 03-031-1B). Normal bronchial/tracheal epithelial Cells (PCS-300-010) were grown in serum-free Airway Epithelial Cell Basal Medium (ATCC, PCS 300-030) supplemented with the appropriate cell growth kit (ATCC, PCS-300-040). All cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.