Dld 1

CRC cell lines DLD-1 and RKO were purchased from ATCC (Manassas, VA). Their respective drug-resistant derivatives, DLD1-DR and RKO-DR were a kind gift from the lab of Zhenghe Wang (CWRU, Cleveland, OH). DLD1-DR cells were developed with resistance to 5ʹ-fluorouracil (5ʹ-FU) (Millipore-Sigma, St. Louis, MO), to an IC₅₀ of 210.6 µM vs IC₅₀ of 2.5 µM for DLD-1 cells, as previously described 55 (link). RKO-DR cells were developed with resistance to combined treatment of 10 µM 5ʹ-FU and 15 µM CB-839, a glutaminase inhibitor (Selleck Chemicals, Houston, TX), as shown in Figure S1A. The DLD-1 and DLD1-DR cells were cultured in McCoy's 5A medium (Thermo Fisher Scientific, Waltham, MA). The RKO and RKO-DR cells were cultured in RPMI-1640 medium (Millipore-Sigma). Both the media were supplemented with 10% fetal bovine serum and 100 Units/mL Penicillin/Streptomycin. All the cells were grown at 37 °C and 5% CO₂.

Three primary colon cancer cell lines (1, 2, and 3) were generous gifts from Prof. Weiping Zou (University of Michigan, USA). Colon cancer cell lines DLD-1, HT29, SW480, SW620, HCT116 and the lymphoma cell line Jurkat were obtained from the American Type Culture Collection (ATCC). DLD-1, SW480, SW620, Jurkat and primary colon cancer cell lines were cultured in RPMI-1640 medium (Gibco) containing 10% foetal bovine serum (FBS) (Science cell) and 1% penicillin/streptomycin. HT29 and HCT116 were cultures in McCoy's 5A medium (Gibco) with 10% FBS and 1% penicillin/streptomycin. Cells were maintained in a humidified incubator with 5% CO₂ atmosphere at 37°C.

Human colon cancer epithelial cells DLD-1 (Male, Dukes’s stage C), HCT116 (Male, Dukes’s stage D), and colonic myofibroblast CCD-18co cells (Female) were obtained from the Korean Cell Line Bank, and SW48 (Female, Dukes’s stage C) was purchased from ATCC. DLD-1 and HCT116 cells were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) and CCD-18co and SW48 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) with L-glutamine (300 mg/L) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 μM). DLD-1 cells were treated with TGFβ (R&D systems, Minneapolis, MN, USA), PDGF-D (R&D systems), imatinib (Tocris Bioscience, Bristol, UK), 2-APB (Tocris), and ML-9 (Sigma Aldrich, St. Louis, MO, USA) for 8 or 16 h. The conditioned medium was obtained from the supernatant of cells collected 8 h after PDGF-D stimulation, and treated with fresh medium in a 1:1 ratio to CCD-18co cells. To prevent mycoplasma contamination, Plasmocin™ (Invitrogen, San Diego, CA, USA, ant-mpp) was added to the medium.

The colorectal cancer cell lines, DLD-1 and LoVo, were purchased from Bioresource Collection and Research Center, Taiwan. DLD-1 cells (Dukes' type C, human colorectal adenocarcinoma) were incubated in high glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). The LoVo cells (Dukes' type C, grade IV, human colorectal adenocarcinoma) were grown in DMEM/F12 medium (Gibco) contained with 10% fetal bovine serum and 1% penicillin/streptomycin. All cultured cells were maintained at 37C and 5% CO2.

Human cancer cell lines HL-60, K562, Jurkat, HeLa, A549, MCF-7, HepG2, A431, and MIA PaCa-2, along with human normal cell lines WI-38 and 1C3D3, were obtained from the RIKEN Cell Bank (RIKEN BRC, Tsukuba, Japan). Human normal cell line YS-1 was obtained from the JCRB Cell Bank (Osaka, Japan). Human cancer cell lines U937, PC-3, DLD-1, Hep3B, WM266-4, SK-MEL-28, HT-1080, and BxPC-3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HL-60, K562, Jurkat, U937, MCF-7, DLD-1, and BxPC-3 cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (Sigma-Aldrich), 50 units/mL penicillin G (Gibco), and 50 μg/mL streptomycin (Gibco). HeLa, A549, PC-3, HepG2, Hep3B, WM266-4, SK-MEL-28, HT-1080, A431, MIA PaCa-2, and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal calf serum, 50 units/mL penicillin G, and 50 μg/mL streptomycin. YS-1 cells were cultured in DMEM containing 5% fetal calf serum, 50 units/mL penicillin G, and 50 μg/mL streptomycin. 1C3D3 cells were cultured in DMEM containing 5% fetal calf serum, 10% newborn bovine serum (SAFC Biosciences, Lenexa, KS, USA), 2.5% horse serum (Gibco), 50 units/mL penicillin G, and 50 μg/mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO₂.