Kyse150

Thirty‐one cases of ESCC samples and paired normal samples were obtained from the First Affiliated Hospital of Zhengzhou University, which was approved by the Research and Ethics Committee of Zhengzhou University. Human ESCC cell lines including Eca109 and KYSE450 and normal oesophageal epithelial cell Het‐1A were purchased from Qingqi Biotechnology Development Co., Ltd (Shanghai, China), and ESCC cell KYSE150 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). The above cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS and double antibiotics including Penicillin and Streptomycin. These cells were placed in an incubator with 5% CO₂ at 37°C.

The esophageal cancer cell lines KYSE30, KYSE150, ECA109, and normal esophageal epithelial cell line HET1A were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). All cell lines were cultured according to the manufacturer's instructions.

Normal human esophagus epithelial cell line Het-1A (IM-H274, Immocell Biotechnology Co.,Ltd., Xiamen, China) and human ESCA cell line TE-1 (CL-0231, Procell Science&Technology Co.,Ltd., Wuhan, China), KYSE30 (CL-0577, Procell), KYSE-150 (CL-0638, Procell) were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, USA), supplemented with 100 U/mL streptomycin and penicillin. Cells were lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). The equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for one hour at room temperature. The membranes were incubated with antibody against BIN3 (TA502145, 1:2000, OriGene Technologies, Wuxi, China), E-cadherin (ab1416, 1:1000, Abcam, Cambridge, UK), N-cadherin (ab76011, 1:5000, Abcam) overnight at 4°C. The membranes were incubated with corresponding secondary antibodies.

The human ESCC cell lines TE‐1 (RRID:CVCL_1759), KYSE‐30 (RRID:CVCL_1351), KYSE‐150 (RRID:CVCL_1348), and KYSE‐170 (RRID:CVCL_1358) were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China) and stored in our laboratory. These cell lines have been authenticated in the past 3 years using short tandem repeat analysis. TE‐1, KYSE‐30, and KYSE‐150 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, whereas KYSE‐170 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat‐inactivated FBS (GIBCO, Thornton, NSW, Australia) in a 37 °C sterile incubator with 5% CO₂. In this study, the cells used in all experiments were mycoplasma‐free.

Four kinds of human esophageal cancer cells inculding TE1, KYSE30, KYSE150 and KYSE170 were chosen to study, which were purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, Hubei, China). There are no mycoplasma contamination among them. All cell lines were culture with RPMI1640 medium (GIBCO, USA) containing with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml streptomycin, which were maintained at 37 °C in a humidified atmosphere of 5% CO₂. For drug treatment, cells were cultured with or without 10 ng/mL recombinant human RANTES (CCL5) (Peprotech, USA) in medium. For transfection, siRNAs were supplied by Guangzhou RiboBio Co.,Lid.( Guangzhou, China). The hsa_circ_0060927 sequence was cloned into the pLC5-ciR vector and synthesized by Geneseed Biotech Co.( Guangzhou, China). According to the manufacturer’s instruction, ESCC cells inoculated in 6-well cell plates (1 × 10⁶) were transfected with pLC5-ciR vector or siRNA by using FuGENE HD transfection Reagent (Promega, USA) or HiperFect transfection Reagent (Promega, USA). 6 h later, the complete medium was used to instead of the transfection medium. After a further culture for 48 h, the cells were collected.