A series of GC cell lines (GSE-1, BGC823, MKN45, MGC803, AGS, MKN28) were obtained from Foleibao Biotechnology Development (Shanghai, China). The cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (NBCS) (PAA Laboratories, Inc., Pasching, Austria). All of these cell lines were incubated in a humidified chamber with 5% CO2 at 37 °C. For inhibitor treatment, 10 mmol/L PI3K inhibitor LY294002 (Cell Signal Technology, Danvers, MA) was added in the cultured cells every two days.
LIFR plasmids, miR-589 mimic, anti-miR-589 oligos and all siRNA oligos including c-Jun specific siRNAs (si-1973, si-1554, si-2358 and si-1113) were purchased from GenePharma (Shanghai, China). GC cells at exponential growth phase were plated into 6-well plates for 24 h at a density of 0.5 × 105 cells/mL, and transfected with 1 mg of siRNA or 4 μg cDNA using Lipofectamine 2000 reagent (Invitrogen; Carlsbad, Calif, USA) in reduced serum medium (OPTI-MEM-I; Invitrogen) according to the manufacturer’s protocol.
LIFR plasmids, miR-589 mimic, anti-miR-589 oligos and all siRNA oligos including c-Jun specific siRNAs (si-1973, si-1554, si-2358 and si-1113) were purchased from GenePharma (Shanghai, China). GC cells at exponential growth phase were plated into 6-well plates for 24 h at a density of 0.5 × 105 cells/mL, and transfected with 1 mg of siRNA or 4 μg cDNA using Lipofectamine 2000 reagent (Invitrogen; Carlsbad, Calif, USA) in reduced serum medium (OPTI-MEM-I; Invitrogen) according to the manufacturer’s protocol.