Sgc 7901

Human GC tumor cell lines SGC-7901 and MKN-45 were purchased from iCell Bioscience (Shanghai, China). The 5-Fu-sensitive gastric cancer line, SGC-7901, was cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (Gibco, Gaithersburg, MD, USA) at 37 °C in a 5% CO₂ incubator. The cells were digested with 0.25% trypsin and routinely passaged when the cells grew to 70–80% confluency. The stepwise addition method was used, starting from addition of 10 nmol/L 5-Fu (Yuanye Bio-Technology, Shanghai, China) until the cells can grew stably in culture medium containing 10 μM 5-Fu; the constructed drug-resistant cell line was named SGC-7901-5-Fu. The drugs were withdrawn 2 weeks before the experiment and routinely cultivated for future use.

Primary antibodies used were rabbit monoclonal anti-Rictor (ab70374, Abcam, Cambridge, MA), anti-Cav 1(Rabbit mAb #3276), Akt (pan) (Rabbit mAb #4691, Phospho-Akt (Ser473) (Rabbit mAb #4060) (Cell Signaling Technology, Shanghai, China), Caspase-3 monoclonal antibody (CPP32-4-1-18), Bcl-2 polyclonal antibody (PA5-11379), Bax monoclonal antibody (6A7), actin monoclonal antibody (ACTN05 (C4) and Biotin (ThermoFisher, Shanghai, China). Rictor plasmid (Addgene #1860) and Cav 1 plasmid (Addgene27703) were purchased from Addgene (USA). Rictor-siRNA and Cav 1-siRNA were purchased from GenePharma Co., Ltd. (Shanghai, China). Ly294002 (#9901) was purchased from Cell Signaling Technology. Human GC cell lines SGC-7901 and AGS were purchased from iCell Bioscience Inc (Shanghai, China) in Feb 2018.