Hek293t

This research used gastric cancer cell lines AGS, HGC27, MKN45, NCI-N87, and SNU-1, bought from Procell Life Science & Technology Co., Ltd. (Wuhan, China); HEK293T and human gastric mucosal epithelial cell line GES-1 were all stored in Dr. Zhou’s laboratory under standard conditions. The HUVEC endothelial cell line was bought from Fu Heng Biology (Shanghai, China). All cell lines were free of mycoplasma contamination. Cells were cultured in RPMI 1640 or ECM medium supplemented with 10% fetal bovine serum (FBS) (Procell, Wuhan, China) and 1% Penicillin/Streptomycin in a cell incubator.
shRNA was purchased from Shandong Virgin Bioscience. The plasmid of ESM1 (with flag label) was purchased from Guan Nan Biotechnology Co., Ltd. (Hangzhou, China). Gastric cancer cells in the logarithmic growth phase were digested with trypsin a day before, resuspended in a complete medium, and counted. An appropriate number of cells were seeded in 6-well plates to ensure that the confluence rate of the cells reached 50% before transfection experiments the next day. After transfection, the 6-well plates were placed in the cell incubator for 48 h. Finally, RT-qPCR and a Western blot were used to detect the overexpression and silencing efficiency of ESM1 mediated by the plasmid and shRNA in gastric cancer cells.

The human HCC cell lines LM3, HepG2, and human embryonic kidney HEK293T cells were purchased from the Procell Life Science&Technology Co, Ltd (Wuhan, China). LM3, HepG2, and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 41965, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, 10270). All cells were cultured at 37 °C, in 5% CO₂ humid atmosphere.

Human embryonic kidney cells HEK-293T (Procell, Wuhan, China) and GC cells MKN45 (Procell) were cultured with RPMI-1640 medium (Thermo Fisher Scientific, Rockford, IL) containing 10% FBS and 1% penicillin-streptomycin sulfate (Thermo Fisher Scientific) in an incubator with 5% CO₂ at 37 °C.

Human vascular smooth muscle cells (T/G HA‐VSMC) and human renal epithelial cells (HEK‐293T) were obtained from Procell company. 10% fetal bovine serum (GIBCO BRL) was added to DMEM (GIBCO, USA), and all cells were incubated under 5% CO₂ at 37℃ in two humidified incubators.

HtNSCs were isolated according to our protocol described previously.¹⁰ Briefly, the hypothalamic tissues of mice were isolated, then the tissues were shredded and digested with 5 ml of TrypLE™ Express enzyme (Gibco) at 37°C for 30 min. One millilitre pre‐cooled neural cell complete medium (Neurobasal medium (Gibco) + 2% B27 (Invitrogen) + 20 ng/ml EGF (Peprotech) + 20 ng/ml bFGF (Peprotech)) were added to digested tissue to terminate digestion. After centrifugation, precipitates were collected and resuspended in a medium, passed through 100‐μm and 40‐μm cell sieves to separate them into single‐cell suspensions. Single‐cell suspension was inoculated in ultralow‐adhesion 6‐well plates at the density of 10⁶ cells per well. The medium was changed every 3 days. Passed through the generations once in about 2 weeks. Experiments were performed with htNSCs that had been passaged to 3–7 generations.
HEK293T was purchased from Procell Co (China). Cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Procell). CAD cell line were cultured in 10% DMEM/F‐12 (Gibco) with 10% FBS and 1% PS. All cells were cultured at 37°C, 5% CO₂, and saturated humidity.

HEK293T, human colonic epithelial cell line NCM460 and human colorectal cancer cell lines SW480, DLD-1, HT-29, RKO, and HCT116 were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China) and the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) respectively. The cells were routinely cultured in the recommended medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃ in a 5% humidified and abacterial incubator.