Kyse410

Human specimens were collected from patients who underwent ESCC resection at the Department of Thoracic Surgery of the Second Affiliated Hospital of Nanchang University. A total of 6 pairs of ESCC specimens and paracancerous specimens were collected. After separation of the samples, some of the esophageal cancer tissues were rapidly frozen in liquid nitrogen and then stored in a −4°C refrigerator to avoid degradation. Normal human esophageal epithelial cell lines (HEEC) from JiNiu Biologicals (China) and TE-1, KYSE-30, KYSE-410, and KYSE-520 cell lines from Wuhan Procell (China) were maintained in DMEM (Gibco, USA) (HyClone, USA) replenished in 10% FBS at 37°C and 95% air with 5% CO2.
RNA was extracted from the cells using TRIzol (Invitrogen, USA) and RNA extraction kits. RNA was converted into cDNA using the PrimeScript RT Reagent Kit (Takara, Japan) and analyzed for gene expression via qRT-PCR. Primers are shown in Supplementary Table 1.

Authenticated ESCC lines TE-1 and KYSE-150 were purchased from the Cell Bank, Type Culture Collection of Chinese Academy of Sciences; KYSE-30 and KYSE-410 were obtained from the Procell Life Science & Technology; EC109 was purchased from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (BI) at 37 °C with 5% CO₂ in a humidified incubator. All experiments were performed with mycoplasma-free cells.
Human ESCC and paired adjacent noncancerous tissues from 75 ESCC patients who underwent esophagectomy without prior chemotherapy or radiotherapy were obtained from the First Affiliated Hospital, Shihezi University School of Medicine. Subjects were recruited with written informed consent and with approval from the Ethics Committee of the First Affiliated Hospital, Shihezi University School of Medicine.

Four ESCC cell lines (KYSE140, KYSE180, KYSE450, and KYSE510) and one immortalized esophageal cell line (NE1) were kindly provided by Professor Li Fu (Department of Pharmacology and International Cancer Center, Shenzhen University). The other four ESCC cell lines KYSE30, KYSE150, KYSE410, and TE-1 were purchased from Procell (Procell Life Science&Technology Co,. Ltd, Wuhan, China). The ESCC cell lines were cultured in RPMI-1640 (Gibco, USA) supplemented with fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). The stable overexpression and knockdown cell lines KYSE30, KYSE410, KYSE510, and TE-1 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1 μg/mL puromycin (Selleck Chemicals, USA). NE-1 was cultured in keratinocyte serum-free medium (K-SFM) and EpiLife at a ratio of 1:1 supplemented with 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL human recombinant epidermal growth factor (EGF). For verteporfin treatment, cells were incubated with 2.5 μM (MCE, USA) for KYSE30 in FBS-free RPMI-1640 containing 0.1% BSA for 6 h before cells were harvested. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. The cell lines were authenticated by short tandem repeat profiling (STR) (Biowing Applied Biotechnology Co. Ltd, Shanghai, China) and to be free of mycoplasma contamination.

Human ESCA cell lines TE-1, KYSE150, and KYSE410 were purchased from Procell (Wuhan, China). These cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% FBS at 37 °C, 5% CO₂. The pcDNA3.1/β3GNT2 plasmid, pcDNA3.1/CREB1 plasmid, empty pcDNA3.1 plasmid, a lentiviral vector expressing β3GNT2 shRNA, a lentiviral vector expressing CREB1 shRNA, control lentiviral vector, miR-133b inhibitor, inhibitor control, miR-133b mimics, and mimics control were constructed by GenePharma (Shanghai, China). All transfections were conducted with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Stable clones were selected by puromycin or G418.