Sgc7901

DMEM containing 10% FBS (Gibco, Thermo Fisher, USA) was used to culture SGC7901 (human GC cell line) and GES-1 (human gastric mucosal epithelial cells, COBIOER, Nanjing, China) in a humid environment with at 37 °C with 5% CO₂.OSBPL1A and its negative control (sh‐NC, 1 μg) were synthesized by Genesee Biotechnology Co., Ltd. The OSBPL1A construct was generated using the pcDNA plasmid from Thermo Fisher Scientific. Lipofectamine 3000 (Invitrogen) was used for cell transfection following the protocol. The transfected cells were then cultured for 48 h prior to conducting the experiment. RT-qPCR.
Extraction of total RNA was realized using TRIzol reagent (Thermo Fisher, USA). RT-qPCR with the use of FastStart Universal SYBR Green Master (Roche, Switzerland) was performed on each sample (2 μg) on a LightCycler 480 PCR System (Roche, USA). The reaction volume of cDNA consisting of 20 μl (appropriate amount of water, 0.5 μl of forward and reverse primers, 10 μl of PCR mixture, 2 μl of cDNA template) served as a template. The PCR cycling was operated with DNA denaturation at 95 °C for 30 s (s), 45 cycles at 94 °C for 15 s, at 56 °C for 30 s, and at 72 °C for 20 s. The threshold cycle (CT) data were standardized to GAPDH by 2^−ΔΔCT. Table 1 listed the sequences of primer pairs for targeted genes.

The GC cell lines SGC7901, BGC823, AGS, MKN45, and SNU-719, and the gastric mucosal epithelial cell line GES1 were purchased from the Cobioer Biosciences Co., Ltd (Nanjing, China). All cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco, Grand Island, NY, USA). Cycloheximide, proteasome inhibitor MG132, and chloroquine diphosphate were purchased from Selleck Chemicals (Houston, TX, USA).

The GC cell lines SGC7901, BGC823, AGS, MKN45, and SNU-719, and the gastric mucosal epithelial cell line GES1 were purchased from the Cobioer Biosciences Co., Ltd (Nanjing, China), which have been tested and authenticated by DNA short tandem repeat test. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) at 37 °C in an incubator of 5% CO₂ and 95% atmosphere.