Kato 3

Three GC cell lines (KATO-III, SNU216, and SNU601) were purchased from the Korean Cell Line Bank and maintained in RPMI1640 medium (Hyclone, Logan UT, USA) supplemented with 10% (v/v) calf serum (Hyclone) at 37 °C in a 5% (v/v) CO₂ humidified atmosphere. The cells were harvested at 300×g for 5 min, incubated in cell-staining buffer containing phycoerythrin (PE)-labeled anti-CD133/1(AC133) antibody (1:10; Miltenyi Biotec, Bisley, UK) for 10 min in a dark refrigerator, and washed with 0.5% (w/v) bovine serum albumin in phosphate-buffered saline, pH 7.2, with 2 mM EDTA. An isotype-matched PE-labeled control antibody (Miltenyi Biotec) was used to label the samples and set gating levels. MoFlo XDP flow cytometry (Beckman Coulter, Brea, CA, USA) was also used to sort cell lines into CD133+ and CD133- populations. The data were analyzed using Summit software, version 5.2 (Beckman Coulter).

Gastric cancer cell lines KATO-III (KCLB No. 30103) and MKN28 (KCLB No. 80102) were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Equitech-Bio, Ingram, TX) supplemented with 100 U/ml penicillin G and 100 μg/ml streptomycin (Invitrogen). Cells were incubated at 37 °C in a humidified atmosphere containing 20% O₂ and 5% CO₂. Type I collagen extracted from rat tail (BD Biosciences, Franklin Lakes, NJ) was dissolved in 0.02 N acetic acid and used for coating tissue culture dishes and inserts for migration assays (5 μg/cm²). Collagen-coated dishes and inserts were washed with PBS immediately before use.
DDR1 inhibitor 3-(2-(pyrazolo[1,5-a]pyrimidin-6-yl)ethynyl)benzamide (7rh benzamide) was dissolved in DMSO to a final stock concentration of 2.5 mg/ml as previously reported [27 (link)].

To validate antibodies for NGF and HO1 used in immunohistochemical staining, we performed western blotting analysis. Four human gastric cancer cell lines (MKN28, MKN45, KATO-III, and NCI-N87) were purchased from the Korean Cell line Bank (KCLB, Seoul, Korea) and cultured in RPMI1640 media. The cells were lysed with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology Inc., Seongnam, Korea). Western blot was performed with primary antibodies for the NGF (Abcam, Cambridge, UK), HO1 (Enzo Life Sciences, Farmingdale, USA), and actin (Sigma, St. Louis, USA).

To validate anti-Ox-PTP and anti-γH2AX antibodies, we performed western blotting analysis using the NCI-N87, MKN45, and KATO-III (Korean Cell Line Bank, Seoul, Korea) human gastric cancer cell line. NCI-N87 and MKN-45 cells were cultured in RPMI1640 media, and KATO-III cells were cultured in IMDM media. To induce oxidative stress, cells were treated with 400 μM H₂O₂ for 24 h. The cells were lysed with PRO-PREP Protein Extraction Solution buffer (iNtRON Biotechnology Inc., Seongnam, Korea) with 1% protease and phosphatase cocktails inhibitor 2,3 (Sigma-Aldrich, St. Louis, MO, USA). The total protein was probed with primary antibodies for Ox-PTP active site (Research and Diagnostic systems Inc., Minnesota, MN), γH2AX (Ser 139) (Cell Signaling Technology, Beverly, MA), and actin (Sigma, St. Louis, MO).

Cell lines were obtained from ATCC (USA) (MKN-28, AGS, KATOIII, HCT-116, MDA-231, SK-BR-3, JIMT-1, U87MG, A549, CEM-119, HeLa, Jurkat and HDF) and from Korean Cell Line Bank (Seoul National University, Korea) (SNU-216, SNU-638, SNU-719, and SNU-C5) were cultured with designated media supplemented with 10% fetal bovine serum and 1x penicillin-streptomycin at 37 °C 5% CO₂ incubator. Human stomach cancer tissues and peri-tumoral normal counter parts were excised within 10 min from the gastrectomy, and preserved in RNAlater solution until RNA purification at 4 °C. All patients provided informed consent prior to collection of tissues, and all methods were performed in accordance with the relevant guidelines and regulations approved by the Institutional Review Board of National Cancer Center of Korea (NCCNCS13732).

Human gastric carcinoma cell lines (NCI-N87, YCC-19, YCC-38, KATOIII, Hs746T, and MKN74), normal gastric epithelial cell line (HFE145), and normal primary human umbilical vein endothelial cell line (HUVEC) were used in this study. NCI-N87 and HUVEC cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, United States). KATOIII, Hs746T, and MKN74 cell lines were purchased from Korean Cell Line Bank (KCLB; Seoul, Korea). YCC-19 and YCC-38 cell lines were established and provided by Sun Young Rha (Yensei University, Seoul, Korea) (Kim et al., 2018 (link)). HFE145 cell line was provided by Hassan Ashktorab (Howard University, MD, United States) (Marlink et al., 2003 (link)). All cell lines were cultured with RPMI 1640 Medium (Hyclone, Logan, UT, United States) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, United States), 100 units/ml penicillin, and 100 μg/ml streptomycin (Hyclone, Logan, UT, United States). HUVECs were maintained in VascuLife EnGS (containing endothelial cell growth supplement; Lifeline Cell Technology, Frederick, MD, United States). All cultured cells were incubated at 37°C in a cell culture incubator with 5% CO₂.