Mkn28

The mixed human gastric tubular adenocarcinoma cell line MKN-28 was obtained from the American Type Culture Collection (Rockville, MD). MKN-28 was cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C in a humidified atmosphere containing 5% CO₂ (18 (link)). MKN-28 cells have previously been reported to be cross-contaminated with MKN-74 cells (19 (link)), however, this had no impact on the outcome of the present study.

GC cell lines HTB-103, AGS, and NCI-N87 used in our study were purchased from American Type Culture Collection (ATCC), and MGC803, MKN45, MKN28, HGC27, and GES-1 cell lines were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All GC cell lines were authenticated by short tandem repeat analysis and had negative results for mycoplasma. HTB-103, AGS, NCI-N87, MKN45, MKN28, and HGC27 were cultured at 37°C in a humidified atmosphere of 5% CO₂ with RPMI-1640 medium (Gibco, San Francisco, CA, USA) containing 10% fetal bovine serum (Gibco) with 100 U/mL penicillin and 100 U/mL streptomycin (Sangon Biotec, Shanghai, China). GES-1 and MGC803 were cultured at 37°C in a humidified atmosphere of 5% CO₂ with DMEM medium (Gibco) containing 10% fetal bovine serum with 100 U/mL penicillin and 100 U/mL streptomycin.
A total of 200 patients in this study underwent D2 gastrectomy at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between 2011 and 2016. All the samples were obtained with the patients’ informed consent and were histologically confirmed. Forty-three pairs of GC tumor tissues and adjacent non-tumor tissues (GC cohort 1) were stored in RNA-latter reagent at −80°C for RNA extraction, and another 157 paired GC tumor tissues and adjacent non-tumor tissues (GC cohort 2) were fixed with formalin and made into tissue microarrays.

The gastric adenocarcinoma cell lines AGS, SNU1, MKN28, MKN45, and STKM2 were used in the study. The immortalized nonneoplastic gastric (GES1) and esophageal (EPC2) cell lines were included as normal controls. AGS and SNU1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). MKN28 and MKN45 cells were obtained from the Riken Cell Bank (Tsukuba, Japan). EPC2 cells were kindly provided by Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA). GES1 cells were kindly provided by Dr. Dawit Kidane-Mulat (University of Texas at Austin, Austin, TX). AGS cells were cultured in Ham's F12 media (GIBCO, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (P/S) (GIBCO). MKN28, MKN45, and GES1 cells were cultured in Roswell Park memorial institute (RPMI) medium (GIBCO) supplemented with 10% FBS and 1% P/S. EPC2 cells were cultured in Keratinocyte serum-free medium supplemented with recombinant epidermal growth factor and bovine pituitary extract (GIBCO). All cell lines were ascertained to conform to the original in vitro morphological characteristics and were authenticated using short tandem repeat (STR) profiling (Genetica DNA Laboratories, Burlington, NC). All cell lines reported here have been tested and had shown to be free of mycoplasma (R&D Systems, Minneapolis, MN).