Human umbilical vein endothelial cells (huvec)

Normal esophageal epithelial cells (HET-1A) were acquired from ATCC (Manassas, VA, USA). TE-1, Eca-109, KYSE150, and HUVEC cell lines were obtained from Procell (Wuhan, China), while KYSE70 cells were provided by TongPai Biotechnology (Shanghai, China). KYSE70 cells and KYSE150 cells were maintained in RPMI-1640 medium, and other cells were cultivated in a DMEM medium. During cell culture, medium was supplemented with 1% penicillin/streptomycin (Procell) and 10% fetal bovine serum (FBS). The culture conditions were 37 °C, 5% CO₂, and 95% humidity.
SiRNAs for circCHSY1 (si-circCHSY1-1 and si-circCHSY1-2), shRNA against circCHSY1 (sh-circCHSY1), miR-1229-3p mimic and inhibitor, Tectonic-1 (TCTN1) overexpressed plasmid (pcDNA-TCTN1) and their negative controls were all provided by GenePharma (Shanghai, China). When the cell aggregation rate reached 80%, 50 nM of miRNA mimic, different substances (20 pmoL of siRNA, 0.8 µg of plasmids, and 100 nM of miRNA inhibitor) were used for cell transfection by Lipofectamine^™ 3000 (Invitrogen, Carlsbad, CA, USA) depending on the experimental requirements.

Human GBM cells U87MG, U251, and HS683 and endothelial cell HUV-EC were obtained from the National Infrastructure of Cell Line Resource in Beijing, China. Cell lines were authenticated at the National Infrastructure of Cell Line Resource, Beijing, China. GBM cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 5% CO₂ and 37 °C. HUV-EC cells were cultured in complete culture medium (Procell Life Science & Technology Co., Ltd., Wuhan, China). Cells were tested mycoplasma free using a Mycoplasma Test Kit (Cellorlab, China). All materials for cell culture were purchased from Gibco Invitrogen (Grand Island, NY, USA) unless otherwise noted.