Te 10

Human normal esophagus epithelial cell line HET1A and human esophageal cancer cell lines ECA-109, EC9706, KYSE150, KYSE450, TE1, and TE10 were bought from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin and 100 μg/ml of streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained in a humidified incubator with 5% CO₂ at 37° C. For hypoxia in vitro model establishment, cells were maintained in conventional DMEM and cultured in a humidity cell incubator at 37° C with atmosphere composition of 2% O₂, 93% N₂, and 5% CO₂ for 24 h.
Patients with esophagus carcinoma were recruited between 2015 and 2019 in Gansu Provincial People's Hospital (Lanzhou City, China). Tumor samples and adjacent normal esophagus epithelial tissue samples (n = 40 pairs) were gathered from the patients who underwent surgical resection in Gansu Provincial People's Hospital. After immediately freezing in liquid nitrogen, resected tissues were stored at −80° C. Written informed consent was obtained from all the patients or their guardians. Ethics clearance was obtained from Institutional Center Ethics Review Committee in Gansu Provincial People's Hospital.

The human esophageal cancer cell lines (Eca109, Eca9706, TE-1, TE-10, TE-11, KYSE140, and KYSE150) were obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (HyClone, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco-BRL), 100 μg/ml streptomycin and 100 units/ml penicillin G (Beyotime Biotechnology, Shanghai, China) in 5% CO₂ and humidified atmosphere at 37 °C. Cells at the third generation of the exponentially growing state were used for all experiments.

The cell lines of EC109, CaES-17, TE-1, TE-10 were obtained from China Center for Type Culture Collection (CCTCC) and the Human normal esophageal epithelial cells (HEEC), HepG2, MKN45, SW60, A549 cell lines were purchased from Beijing Beina Science and technology company. The cells were cultured in DMEM (Gibco, USA) medium supplemented with 15% FBS (Gibco, USA), 100 U/mL of penicillin and 100 mg/mL of streptomycin (Hyclone, USA) at 37 °C in a humidified culture chamber (NAPCO5410, USA) supplied with 5% CO₂ atmosphere.