Nci h2087

Human adenocarcinoma cell lines NCI-H1437, NCI-H1792, and NCI-H2087 and human embryonic kidney HEK293 cells were purchased from American Type Culture Collection (Manassas, VA, USA), and the CL1-5 cells were kindly provided by Dr. Pan-Chyr Yang of National Taiwan University. NCI-H1437, NCI–H1792, NCI-H2087, and CL1-5 cells were maintained at 37°C, in a 5% CO₂ humidified atmosphere, in complete RPMI1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). HEK293 was cultured in Eagle’s minimum essential medium with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA). To obtain CL1-5-conditioned media (CM), the cells were seeded as 1 × 10⁶ cells/100-mm dish and cultivated in normoxic or hypoxic conditions for 24 hr, and the supernatants (CM) were harvested after 24 hr of incubation. Pharmacological inhibitors AG490 and LY294002 (Calbiochem, Nottingham, UK) were used at a concentration (1 μM) that did not affect cell viability of macrophages. WST-1 was used to assess cancer cell viability. All cells tested negative for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza) and were authenticated by short tandem repeats (Promega).

NSCLC cell lines A549, H1299, H1650, and NCI-H2087, and the normal bronchial epithelial cell line 16HBE were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, and maintained in a humidified atmosphere at 37°C with 5% CO₂.