Hgc 27

Five human GC cell lines (MKN74, NCI-N87, MKN-45, HGC-27, and AGS) from the American Type Culture Collection (ATCC; Manassas, VA) were cultivated in the DMEM with 1% Pen/Strep solution and 10% FBS in 5% CO₂ at 37 °C. Insulin like growth factor 1 (IGF-1; 100 ng/mL), the PI3K/AKT pathway activator, was procured from Sigma-Aldrich (St. Louis, MO) to treat HGC-27 and AGS cells.

Human colorectal adenocarcinoma cells (CaCo-2), metastatic SW-620 colon cancer cells, hepatocellular carcinoma cell lines (Hep-3B, HLF and HLE cells), liver hepatoma cells (PLC-5), and gastric carcinoma (derived from metastatic site) N-87 cells were purchased from ATCC. Human gastric carcinoma (derived from metastatic site) cell line (HGC-27) was purchased from Sigma-Aldrich. Human intrahepatic cholangiocellular carcinoma cell line (HUCCT-1) was purchased from JCRB Cell Bank derived from ascitic fluid. All cell lines were cultivated in according to retailer protocols. Briefly, colon cancer cell lines (CaCo-2 and SW-620) were cultured in 10% of Inactivated Fetal Bovine Serum (FBS) exosomes free (Euroclone) in Dulbecco’s Modified Eagle’s Medium (DMEM) with sodium pyruvate, 4.5 g/L glucose (GIBCO), 4mM L-Glutamine (GIBCO) and 5 mL Pen-Strep (penicillin 10,000 u/mL, streptomycin 10,000 u/mL, Lonza Biowhittaker). Gastric (N-87 and HGC-27), hepatocellular carcinoma (Hep-3B, HLE and HLF), hepatoma (PLC-5), and intrahepatic cholangiocellular carcinoma (HUCCT-1) cell lines were cultured with Roswell Park Memorial Institute (RPMI) medium (GIBCO) supplemented with 10% of FBS exosomes free, sodium pyruvate, 4.5 g/L glucose, 4mM L-Glutamine, and 5 mL Pen-Strep.Cells were grown until reaching semi-confluence, in a humidified incubator at 37 °C with an atmosphere containing 5% of CO₂.

The human GC cell lines HGC27 and AGS were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and the human GC cell line MGC803 and human gastric epithelium cell line GES-1 were obtained from the Key Laboratory for Tumor Precision Medicine of Shaanxi Province (Xi’an, China). HGC27 and MGC803 cells were cultured in the RPMI 1640 medium (Sigma-Aldrich, WI, USA), AGS cells were maintained in Ham’s/F-12 medium (Procell, Wuhan, China), and GES-1 cells were grown in Eagle’s minimum essential medium (DMEM, Sigma-Aldrich, WI, USA), containing 10% fetal bovine serum (Biological Industries, Israel) and 1% penicillin/streptomycin (New Cell & Molecular Biotech, Suzhou, China). All cells were incubated in a water-saturated atmosphere of 5% CO₂ at 37 °C and were collected at the peak of the logarithmic growth phase for experiments.

HGC27 (Sigma-Aldrich) and PC-3 (ATCC) cells were cultured according to the manufacturer’s recommendations. The non-targeting and the CSMD3 and ZFHX4 siRNAs were obtained from OriGene (Rockville, MD). The siRNA sequences are as follow: si-CSMD3-1, GGUAUAUUACGAAGAAUUGCAGAGT; si-CSMD3-2, ACAAAUGGAGGAAUACUAACAACAG; si-ZFHX4-1, CGAUGCUUCAGAAACAAAGGAAGAC; si-ZFHX4-2, GGAACGACAGAGAAAUAAAGAUUCA. The siRNAs were transfected into cells using DharmaFECT transfection reagents for 48 hrs according to the manufacturer’s instructions.

One normal human gastric epithelial mucosa cell line (GES-1) was obtained from the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences (Beijing, China). Five GC cell lines AGS (CRL-1739) and SNU-1 (CRL-5971) were primary purchased from the American Type Culture Collection (Manassas, VA, USA); HGC-27(94042256) was from European Collection of Authenticated Cell Cultures (Public Health England; Porton Down, Salisbury, UK); MKN74 (JCRB0255) and MKN1 (JCRB0252) were from Japanese Collection of Research Bioresources Cell Bank (Ibaraki city, Osaka, Japan). All of these cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco, Gaithersburg, MD, USA) medium plus 10% Fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C. AGS and HGC-27 cells were exposed to Ly294002 (10 μM, Sigma-Aldrich) or Rapamycin (100 nM) for 1 day.