Nci h1299

Human NSCLC cell lines A549 (KCLB No. 10185), NCI-H1299 (KCLB No. 25803), NCI-H1650 (KCLB No. 91650), and HCC827 (KCLB No. 70827) were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). The identity of each cell line was confirmed by STR profiling: A549 (D3S1358: 16; vWA: 14; FGA: 23; Amelogenin: X,Y; TH01: 8,9.3; TPOX: 8,11; CSF1PO: 10,12; D5S818: 11; D13S317: 11; D7S820: 8,11), NCI-H1299 (D3S1358: 17; vWA: 16,18; FGA: 20; Amelogenin: X; TH01: 6,9.3; TPOX: 8; CSF1PO: 12; D5S818: 11; D13S317: 12; D7S820: 10), NCI-H1650 (D3S1358: 18; vWA: 18; FGA: 20,23.2; Amelogenin: X; TH01: 9.3; TPOX: 11; CSF1PO: 11; D5S818: 11; D13S317: 11; D7S820: 8,9), and HCC827 (D3S1358: OL; vWA: 18; FGA: 22,24; Amelogenin: X; TH01: 6; TPOX: 8; CSF1PO: 11; D5S818: 12; D13S317: 9; D7S820: 11,12). CSCs were propagated from NSCLC cell lines as tumorspheres under CSC-selective culture conditions, and their stem properties were confirmed as previously described [23 (link),24 (link),25 (link)]. NSCLC tumorsphere cells were cultured in serum-free DMEM/F12 containing 20 ng/mL EGF, 20 ng/mL bFGF, 1 × B-27, 5 μg/mL heparin, 2 mM L-glutamine, and 1% penicillin/streptomycin. Tumorspheres were subcultured through dissociation with Accutase. The cells were maintained at 37 °C in a humidified CO₂ incubator with 5% CO₂ (Thermo Scientific, Vantaa, Finland).

A549 (p53-wild type, p16-null) and A427 (p53-wild type, p16-null) non-small cell lung cancer cells were obtained from the American Type Culture Collection (ATCC, Lot No. 58314291 and 58696830, respectively) and maintained in Dulbecco's modified Eagle's medium (DMEM, Welgene Inc.). NCI-H1299 (p53-null, p16-deficient) cells were obtained from the Korean Cell Line Bank (KCLB) and maintained in Roswell Park Memorial Institute (RPMI) 1640. All media were supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B. Cells were incubated at 37 °C in 5% CO₂ incubator.

All lung cancer cell lines, including A549 and NCI-H1299 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin/streptomycin (Gibco) according to the provider’s recommendations. Human umbilical vein endothelial cells (HUVECs; Lonza, Basel, Switzerland) were maintained in endothelial growth medium-2 (EGM-2; Lonza). All cells were maintained at 37 °C in a humidified incubator with 5% CO₂ (Panasonic Healthcare Company, Tokyo, Japan). Throughout this study, all lung cancer cell lines and HUVECs were passaged less than five times and were used within 2 weeks of thawing. Expi293F™ cells (Gibco) were cultured in Expi293™ expression medium (Gibco) in a humidified Multitron incubator shaker (Infors HT, Basel, Switzerland) at 37 °C with 8% CO₂.

The human lung carcinoma cell lines A549 and NCI-H1299 were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI 1640 (HyClone, South Logan, UT, USA) supplemented with 10% inactivated fetal bovine serum (HyClone) at 37 °C in an atmosphere of 5% CO₂.
The LAB-EHY100 (OncoTherm, Budapest, Hungary) device was used for the hyperthermia treatment. The cells were placed in the heating chamber (LAB-EHY in vitro applicator) with culture medium at 42 °C for 30 min. Irradiation of cells was performed using a ¹³⁷Cs gamma-irradiator (MK 1-68, JL Shepherd, San Fernando, CA, USA) at a rate of 2.75 Gy/min.
The A549 and NCI-H1299 cells were plated into 60-mm dishes and exposed to 0, 2, 4, and 8 Gy radiation. For the combination treatment, cells were treated using the LAB-EHY100 at 42 °C for 30 min prior to irradiation. After 14 days of incubation, the colonies were stained with crystal violet, and those with more than 50 cells were counted. The plating efficiency of the control group and the surviving fraction of each treatment group were calculated. All experiments were performed in triplicate. The cell survival curve was fitted to the  LQ model for the calculation of radiobiological parameters.

In this experiment, we used lung cancer cell lines (HCC827, NCI–H358, NCI–H522, NCI– H1299, NCI– H460, and NCI–H226) and HeLa cell line. We purchased the HCC827, NCI–H358, NCI–H522, NCI– H1299, NCI– H460, and HeLa from the Korea Cell Line Bank, and we purchased the NCI–H226 from the American Type Culture Collection. Lung cancer cells (HCC827, NCI–H358, NCI–H522, NCI– H1299, NCI– H460, and NCI–H226) and HeLa cells were maintained in an RPMI 1640 medium (GIBCO BRL, Rockville, MD, USA) with 10% fetal bovine serum and antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin).

For our study, A549 and NCI-H1299 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, South Korea). CHO-A1, Hela-A2A, and HEK-293T-A2B (Euroscreen, Gosselies, Belgium) were also used in this study. The cell culture medium (Roswell Park Memorial Institute (RPMI) 1640, Thermo Fisher Scientific, Waltham, MA, USA) contained 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA) and was used in accordance with guidelines provided by KCLB. The cells were cultured at 37 °C in an incubator with 5% CO₂. When the cell density reached 90%, subcultures were generated using a trypsin-EDTA solution.