EGFR-Mutant NSCLC Cell Lines and HUVEC Culture

The NSCLC cell lines A549 (ATCC CCL-185), H460 (ATCC HTB-177), H1650 (ATCC CRL-5883) and H1975 (ATCC CRL-5908) were purchased from American Type Culture Collection (Manassas, VA, USA). H1650 and H1975 cell lines have EGFR mutations (delE746-A750 for H1650; L858R and T790 M for H1975) (14 (link)). Human umbilical vein endothelial cells (HUVECs; H-UV001) were purchased from Bioresource Collection and Research Center (Hsinchu City, Taiwan). Cells were grown in complete growth medium [Dulbecco's modified Eagle's medium (Lonza, Basel, Switzerland) for A549 and H460 cells; RPMI-1640 media (Lonza) for H1650 and H1975 cells] supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 30 ng/ml EGF (Invitrogen; Thermo Fisher Scientific, Inc.), 10 U/ml penicillin and 10 µg/ml streptomycin at 37°C and 5% CO₂. Cells were incubated for 72 h and the supernatant of growth medium was collected for detection of VEGF levels. HUVECs were grown in 90% Medium 199 (Lonza) with 25 U/ml heparin (Lonza) and 30 µg/ml endothelial cell growth supplement (Lonza), adjusted to contain 1.5 g/l sodium bicarbonate, 10 U/ml penicillin and 10 µg/ml streptomycin, as well as 10% fetal calf serum. Gefitinib and G418 were purchased from Sigma Aldrich (EMD Millipore, Billerica, MA, USA).