H1299

PC3 (human prostate cancer), H1299 (human non-small cell lung cancer), and SKOV3(human ovarian cancer) cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). PC3, H1299 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and SKOV3 was cultured in RPMI1640, supplemented with 10% heat-inactivated FBS. All cells were grown in a humidified atmosphere constituting 5% CO₂ at 37 °C. ZEB1, NEDD8, HIF1A, and control siRNAs were purchased from M. Biotech (Hanam-si, Gyeonggi-do, South Korea). siRNA sequences are summarized in Supplementary Table S2. The siRNAs were transfected into PC3, H1299, and SKOV3 cells using Lipofectamine RNAiMAX (Invitrogen). The cells were incubated for 2 days and passaged for further experiments.

Human lung ADC cell lines were obtained from the American Type Culture Collection (H1792) or the Korean Cell Line Bank (H23, H358, H1299, H1703, H1792, H1975, HCC1171, HCC2108, and SK-LU-1). All cells were grown at 37°C with 5% CO₂ in RPMI-1640 or Dulbecco's modified Eagle's medium (HyClone) containing 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin (HyClone). All cells were monitored for Mycoplasma contamination using a PCR-based method for detection. Detected Mycoplasma were eliminated using BM cyclins (Roche).

Non–small cell lung cancer H1299 and A549 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Human embryonic kidney 293T (HEK 293T) cells and lung normal epithelial HBE4 cells were acquired from the American Type Culture Collection (Manassas, VA, USA). HBE4 cells were cultured in the keratinocyte serum-free (KSF) medium (Gibco), H1299 and A549 cells were cultivated in RPMI 1640 (Invitrogen), and HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% of foetal bovine serum (Hyclone). All cell cultures were maintained at 37 °C and 5% of CO₂. Transient transfection procedures were performed using the TransFectin™ Lipid Reagent (Bio-Rad Laboratories Inc.) or a Neon^® electroporation device (Invitrogen).

The human NSCLC cell lines H1299 and Calu-1 were purchased from the Korea Cell Line Bank (KCLB) (Seoul, Korea). Cells were maintained according to instructions provided by the KCLB. Recombinant human Shh N-terminal peptide (N-Shh) was purchased from R&D Systems (Minneapolis, MN, USA), and cyclopamine-KAAD was purchased from Calbiochem (San Diego, CA, USA).

Human lung fibroblast IMR90 (KCLB No. 10186), bovine pulmonary epithelium CPAE (KCLB No. 10209), NSCLC A549 (KCLB No. 10185), H1650 (KCLB No. 91650), H1299 (KCLB No. 91299), and H358 (KCLB No. 25807) cell lines were obtained from Korean Cell Line Bank. All NSCLC and CPAE cells were maintained in RPMI 1640 medium (Corning, United States) and IMR90 cells in Minimum Essential Medium (MEM; Corning) containing 10% heat-inactivated FBS and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). All cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.
To assess the cell viability, cells (5 × 10³ cells/well) were seeded in 96-well plates for 24 h. After incubation, gefitinib or cilengitide were added for 24–72 h in culture media containing 10% FBS. Cell viability was assessed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Japan) according to the manufacturer’s instructions. The absorbance was measured by a Multiscan^TM FC microplate photometer (Thermo Fisher Scientific, United States). Cell viability is presented as % of control (untreated cells). Experiments were performed in triplicate.