Kato 3

The human GC cell lines KATO-III, MKN28, SGC7901, AGS, MKN45 and MGC803 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). KATO-III cells were maintained in 80 % IMDM (ATCC, USA) supplemented with 20 % fetal bovine serum (FBS) (Gibco). The other cell lines including MKN28, SGC7901, AGS, MKN45 and MGC803 were cultured in 90 % RPMI-1640 (Gibco) supplemented with 10 % FBS (Gibco). All cells were maintained in a humidified atmosphere of 37 ˚C containing 5 % CO 2 .

Five human GC cell lines were used in this study (HGC27 and NCI-N87 were purchased from ATCC, Manassas, VA, USA; AGS, KATO III, and SNU-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China). All the five cell lines were cytogenetically tested and authenticated before the cells were frozen. Cell viability assays mainly included MTT assay, foci formation assay and anchorage-independent colonyformation assay. For the MTT assay, the cells were seeded at 5 × 10 3 cells/well in 96-well plates. After incubation for 0, 24, 48, and 72 h, 20 μL MTT was added, and the plates were incubated for 2 h in a CO 2 incubator, after which 100 μL dimethyl sulfoxide (DMSO) was added to check the OD value at a wavelength of 570 nm. For the foci-formation assay, 6 × 10 2 cells/well were seeded into 6-well plates and then stained with crystal violet after 10-14 days, depending on different cell lines. For the anchorage-independent colony-formation assay, 8 × 10 3 cells/well were seeded into 6-well plates and incubated for approximately 2 weeks, after which photos were acquired. The specific details of these assays have been previously described [14] .