Az521

Human gastric cancer cell lines, AGS (ATCC), AZ521, MKN45, KatoIII, (RIKEN, BioResource Center, Tsukuba, Japan), SNU216, SNU484, SNU601, SNU638, SNU668 and SNU719 (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI1640 supplemented with 10% FBS. The cell proliferation rate was examined using the Alamar Blue Cell Viability Reagent (Invitrogen, Carlsbad, CA, USA). For the soft agar colony formation assay, cells were suspended in 0.33% agarose contained in the medium and seeded on 0.5% bottom agar. After 21 days of culture, soft agar was stained with Giemsa solution (Wako, Osaka, Japan) and colony numbers were scored.
Cells were transfected with pcDNA3, pcDNA-Flag-RUNX3 or pcDNA-Flag-RUNX3(R122C) vector.(6 (link)) KatoIII-R3 stable cell line was constructed by transfection with pcDNA-RUNX3 and selected with G418 (Wako) at 100 μg/mL. To knock down gene expression, cells were transfected with Silencer Select siRNA for RUNX3 or β-catenin (Ambion, Cambridge, MA, USA).
To examine the Wnt activation level, cells were cotransfected with super 8× TOPflash or Super 8× FOPflash (Addgene, Cambridge, MA, USA), together with pcDNA3, pcDNA-Flag-RUNX3 or pcDNA-Flag-RUNX3(R122C).(6 (link)) At 24 h after transfection, the luciferase activity was measured using a Luciferase assay system (Promega, Madison, WI, USA).

The human gastric cancer cell lines used, that is, HSC-39, HSC-43, HSC-59, HSC-60, HSC-64, HSC-44PE, 58As9, 58As1, 44As3 and 44As3Luc, have been described previously.31 (link)–34 (link) MKN1, MKN7, MKN74, NUGC-4, KATO-III, MKN45 and IM95 were obtained from the Health Science Research Resources Bank. AGS, NCI-N87 and SNU-5 were obtained from the American Type Culture Collection (ATCC). GCIY, ECC12, AZ521 and KE-97 were provided by the RIKEN Bio-Resource Center through the National Bio-Resource Project of the MEXT, Japan. These cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 10 units/mL of penicillin and 10 μg/mL of streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.