Met 5a

LN229, A431, SK-BR-3, Chinese hamster ovary (CHO)-K1, HEK-293T, Met-5A, and P3U1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). LN229/HER2 and CHO/HER2 were produced by transfecting pCAG/PA-HER2-RAP-MAP^{(14 (link))} into LN229 and CHO-K1 cells using Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA) and Lipofectamine LTX (Thermo Fisher Scientific, Inc.), respectively. A few days after transfection, PA tag-positive cells^{(15 (link))} were sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan).
CHO-K1, CHO/HER2, and P3U1 cell lines were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN229, LN229/HER2ec, LN229/HER2, A431, SK-BR-3, HEK-293T, and Met-5A cell lines were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air.

The human GC cell lines, NUGC4 (RCB1939), MKN7 (RCB0999), HGC27 (RCB0500), MKN28 (RCB1000), MKN45 (RCB1001), and MKN74 (RCB1002) and the immortalized fibroblast cell line, WI-38, were obtained from the RIKEN BioResource Research Center Cell Bank (Tsukuba, Japan). The immortalized human mesothelial cell line, Met-5A, was purchased from the American Type Culture Collection (Rockville, MD, USA), and the human umbilical vein endothelial cell line, HUVEC from PromoCell (Heidelberg, Germany).
WI-38, Met-5A, and all GC cell lines besides HGC27 were cultured in Roswell Park Memorial Institute medium (RPMI, Nacalai Tesque, Kyoto, Japan) with 10% fetal bovine serum (FBS, System Biosciences, Palo Alto, CA, USA), 100 µg/mL streptomycin, and 100 U/mL penicillin. HGC27 was maintained in Dulbecco’s Modified Eagle Medium including the same amount of FBS and antibiotics. HUVEC were cultured in endothelial basal medium (Lonza, Allendale, NJ, USA) with the endothelial growth supplement SingleQuots (EGM-2; Lonza). All cells were cultured in a humidified 5% carbon dioxide incubator at 37 °C.

The human ESCC cell lines TE2, TE5, TE8, TE11, TE13, and TE15, and the immortalized fibroblast cell line WI-38, were obtained from the RIKEN BioResource Research Center Cell Bank (Tsukuba, Japan). The human ESCC cell lines KYSE70, KYSE150, and KYSE170 were obtained from the Japanese Collection of Research Bioresources Cell Bank Center (Osaka, Japan). The immortalized human esophageal mucosal cell line Het-1A and immortalized human mesothelial cell line Met-5A were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The human umbilical vein endothelial cell (HUVEC) line was purchased from PromoCell (Heidelberg, Germany). ESCC, WI-38, Het-1A, and Met-5A cell lines were cultured in Roswell Park Memorial Institute (RPMI) medium (Nacalai Tesque, Kyoto, Japan) mixed with 10% fetal bovine serum (FBS; System Biosciences, Palo Alto, CA, USA), 100 µg/mL streptomycin, and 100 U/mL penicillin. HUVEC were cultured in endothelial basal medium (EBM; Lonza, Allendale, NJ, USA) with the endothelial growth supplement SingleQuots (EGM-2; Lonza). All cells were cultured at 37°C in a humidified 5% carbon dioxide incubator.