Mkn74

The human GC cell lines MKN7, MKN45, MKN74, HGC27, and NUGC4 were obtained from the Riken Cell Bank (Tsukuba, Japan). These cell lines were cultured in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 100 µg/ml of streptomycin, 100 U/ml of penicillin (Nacalai Tesque), and 10% fetal bovine serum (FBS) (Sigma-Aldrich Japan, Tokyo, Japan) in a humidified incubator at 37 °C in 5% CO 2 . The rabbit polyclonal anti-LRRC8A antibody used in the immunohistochemical (IHC) analysis was purchased from Abcam (ab-157489, Cambridge, MA, UK). The mouse monoclonal anti-LRRC8A antibody used in western blotting was purchased from Sigma-Aldrich (SAB1412855 St. Louis, MO, USA). A mouse monoclonal anti-beta-actin (ACTB) antibody was purchased from Sigma-Aldrich (A5441). Rabbit polyclonal anti-SAPK/JNK, anti-phospho-SAPK/JUN, and anti-phospho-p53 (Thr81) antibodies, a mouse monoclonal anti-p53 (1C12) antibody, and a rabbit anti-p21 (Waf1/ Cip1) antibody were purchased from Cell Signaling Technology (anti-SAPK/JNK; #9252/anti-phospho-SAPK/JNK; #9251/anti-phospho-p53 (Thr81); #2676/anti-p53 (1C12); #2524/anti-p21 (Waf1/Cip1); #2947, Beverly, MA, USA). Anti-horseradish peroxidase (HRP)-linked anti-mouse and anti-rabbit secondary antibodies were obtained from Cell Signaling Technology (anti-mouse; 7076S/anti-rabbit; 7074S).

The human GC cell lines KATOIII, HGC27, NUGC4, MKN45, and MKN74 and fibroblast cell line WI-38 were obtained from the Riken Cell Bank (Tsukuba, Japan). The human normal mesothelial cell line MeT-5A (CRL-9444) was purchased from ATCC (Manassas, VA, USA). These cells were grown in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% fetal bovine serum (FBS). Cells were cultured in flasks or dishes in a humidified incubator at 37 °C under 5% CO 2 in air.
The monoclonal anti-NIS antibody used in the immunohistochemical analysis was obtained from Thermo Fisher Scientific (Massachusetts, USA). The rabbit monoclonal c-Jun N-terminal kinase (JNK), phosphorylated JNK, extracellular signal-regulated kinase (ERK), phosphorylated ERK, p38, phosphorylated p38, NF-κB, phospho-NF-κB, and cleaved caspase 3 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibodies against GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-rabbit or mouse secondary antibodies was purchased from Cell Signaling Technology (Beverly, MA). Human Stomach Total RNA was purchased from Clontech Laboratories, Inc (Mountain View, CA).

This study was approved by the institutional ethics committee of the National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Japan. Written informed consent was obtained by the commercial source (bioChain, Hayward, CA, USA).
Cell culture. The GC cell lines MKN45 and MKN74 were purchased from RIKEN Cell bank (Tsukuba, Japan). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FbS) (Life Technologies, Grand Island, NY, USA). Cell lines were cultured with 5% carbon dioxide at 37˚C in a humidified chamber. shRNA transfection. Cells were plated in 6-well plates (Asahi Techno Glass, Tokyo, Japan) and cultured until they reached 80% confluence, then transfected and cultured for an additional 48 h. Fz2 shRNA (OriGene, Rockville, MD, USA) was transfected into cells by using Lipofectamine LTX (Life Technologies), according to the manufacturer's instructions. Briefly, shRNA was incubated with PLUS reagent for 5 min, after which LTX reagent was added. A 15-min incubation at room temperature ensued, and the complex was subsequently applied to the cell culture medium. An shRNA negative control was also purchased from OriGene.