Mkn45

Human gastric cell lines included AGS, MKN28, MKN45, and GES1 cells. The MKN28 and MKN45 were purchased from Riken Cell Bank (Tsukuba, Japan). AGS and cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA), whereas GES1 Human gastric epithelial cell line was a gift from Dr. Kidane-Mulat (University of Texas-Austin). Cells were cultured Following the recommended culture medium, RPMI 1640 medium (Thermofisher Scientific, USA) or F12 Ham medium (Thermofisher Scientific, USA), with 10 % fetal bovine serum (Thermofisher Scientific, USA) and 1 % penicillin/streptomycin (Thermofisher Scientific, USA). In addition, the recombinant human fibroblast growth factor 19 (FGF19) was purchased from Thermofisher Scientific (Thermofisher Scientific, USA).

Six human gastric carcinoma cell lines, H-111-TC, HGC-27, Kato-III, MKN-1, MKN-45 and NU-GC-4 were purchased from Riken Cell Bank (Tsukuba, Japan) and used in the present study. According to the description by the Riken Cell Bank and literature (22 (link)–27 (link)), these cell lines originated from 1 unknown origin (H-111-TC) and 5 metastatic carcinoma, as well as 2 tubular (well differentiated or adenosquamous) carcinoma (H-111-TC and MKN-1) and 4 poorly differentiated carcinoma including signet ring cell type. HGC-27 and MKN-1 cell lines were maintained in Dulbeccos modified Eagles medium (DMEM; Gibco-BRL, Rockville, MD, USA), whereas NU-GC-4, Kato-III, MKN-45 and H-111-TC cell lines were maintained in RPMI-1640 (Gibco-BRL), supplemented with 10% fetal calf serum (FCS), penicillin, and streptomycin, at 37°C in a humidified atmosphere of 5% CO₂. When they reached 80–90% confluence, the cells were washed with phosphate-buffered saline (PBS) and homogenized immediately in Isogen reagent (Nippon Gene, Osaka, Japan). Total RNA was extracted according to the manufacturers instructions.

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).

MKN7, MKN45, MKN74, HGC27, and NUGC4 human GC cell lines were purchased from the Riken Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) containing 100 μg/mL of streptomycin, 100 U/mL penicillin, and 10% FBS at 37 °C in a 5% CO₂ incubator. Rabbit polyclonal anti-ANO5 antibody was obtained from Funakoshi (GTX81161) for immunohistochemical (IHC) analysis and western blotting. Mouse monoclonal anti-β-actin antibody was provided by Sigma-Aldrich (St. Louis, MO, United States) and HRP-conjugated anti-rabbit and mouse secondary antibodies by Cell Signaling Technology (Beverly, MA, United States).