Mkn74

The gastric cancer cell lines MKN74, Kato-III, SH-10-TC (Riken Bioresource Center, Tsukuba, Japan), AGS (ATCC), SNU216, SNU484, SNU601, SNU638, SNU668, and SNU719 (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI1640 or DMEM supplemented with 10% FBS. All cell lines were authenticated by an isoenzyme analysis or short tandem repeat analysis and initially expanded and cryopreserved within one month of receipt. Cells were used within three months after thawing frozen vials. The construction of WT-ER FOXO3- and Act-ER FOXO3-expressing cells is indicated in Supplementary Information.
For the colony formation assay, 2 × 10³ cells were cultured in a 6-well plate in the presence or absence of tamoxifen (Sigma-Aldrich) at 1 μM for 10 days. Cells were then stained with Giemsa solution, and the colony numbers per well were counted. For the cell proliferation analysis, 1 × 10³ cells were seeded in a 96-well plate, and the cell viability was examined with a Cell Titer-Glo Cell Viability Assay (Promega, Madison, WI). The luciferase activity was measured by a Centros XS³ LB960 (Berthold Technologies, Bad Wildbad, Germany). All cell culture experiments were repeated three times. Mycoplasma testing was performed using a direct immunofluorescence test.

Human gastric cancer cell lines (KATOIII, SNU16, AGS, NCI-N87, Hs746T) and human colon cancer cell lines (HCT116, HT-29) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Other human gastric cancer cell lines (MKN1, MKN7, MKN45, MKN74, HGC-27, GCIY) were obtained from the Cell Bank, RIKEN BioResource Center (Kyoto, Japan). Another human gastric cancer cell line (FU97) was obtained from the Health Science Research Resources Bank (Japan), and ISt-1 was a gift from Dr. Masanori Terashima. All cell lines were tested and free of mycoplasma contamination. The Dearing strain of reovirus serotype 3 (a gift from P. Lee, Dalhousie University, Halifax, NS, Canada) was propagated in suspension cultures of L929 cells (from ATCC) and purified according to previously established methods [8 (link),9 (link)] with the exception that β-mercaptoethanol was omitted from the extraction buffer. Viral titers were also established using L929 cells [9 (link)].

The AGS cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MKN74, MKN1, and GCIY cell lines were purchased from RIKEN BioResource Center (Ibaraki, Japan). To prepare the acidic culture medium (pH 6.5), Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Sigma-Aldrich, MO, USA) was supplemented with 10 mM PIPES. For the preparation of control culture medium (pH 8.0), DMEM with high glucose and NaHCO₃ (FUJIFILM Wako, Osaka, Japan) was supplemented with 10 mM HEPES. These media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific) and were adjusted to their respective pH at 37 °C. The pH of the medium was monitored before and after incubation to confirm that it remained constant throughout the experiment.

The human gastric cancer cell line AGS (ATCC CRL-1739; gastric adenocarcinoma) was purchased from the American Type Culture Collection (Manassas, VA, USA). Human gastric cancer cell line MKN-74 was obtained from RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). Human gastric cancer cell lines MKN-1 and SNU-668 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). These cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBCO-BRL, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The cells were cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO₂.
The normal rat gastric epithelial cell line RGM-1 was obtained from RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). RGM-1 cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM; GIBCO-BRL, Scotland, UK) and Ham’s F-12 medium (GIBCO-BRL), supplemented with 10% fetal bovine serum (GIBCO-BRL, Scotland, UK) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The cells were cultured at 37 °C in a humidified atmosphere containing 95% air and 5% CO₂.

Human gastric cancer cell lines (MKN7, MKN45, MKN74, KATOIII, and NUGC3) were purchased from the Riken Bioresource Center (Cell Bank, Ibaraki, Japan) and the Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan). All cell lines were cultured in culture medium (RPMI 1640 medium [Sigma-Aldrich, St. Louis, MO] containing 10% heat-inactivated fetal bovine serum [FBS, Life Technologies, Carlsbad, CA], 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B [Antibiotic-Antimycotic; Life Technologies]) at 37°C in 5% CO₂ with 95% humidity.

To investigate the molecular role of FARP1 in the development of gastric cancer, we used four gastric cancer cell lines. The human gastric cancer cell lines MKN7 (RCB Cat# RCB0999, RRID: CVCL_1417), MKN45 (RCB Cat# RCB1002, RRID: CVCL_2791), MKN74 (RCB Cat# RCB1001, RRID: CVCL_0434), and GSU (RCB Cat# RCB2278, RRID: CVCL_8877) were obtained from RIKEN BioResource Center (Tsukuba, Japan). The cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with antibiotics (100 U/mL penicillin) and 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). All cancer cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO₂.