Ccd 18co cells

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 human colorectal adenocarcinoma SW480 cell line and normal colon fibroblast cell line CCD-18Co cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Mouse embryonic fibroblast (MEF) was prepared from embryos (day 12.5) of C57BL/6 mouse as described previously35 (link). The HCT116 human colorectal carcinoma cell line, 293 T cell line, and Wnt3a-secreting L cell line (L-Wnt3a) were purchased from the American Type Culture Collection (Manassas, VA, USA). The HEK293 reporter cell line (stably transfected with TOPFlash, a synthetic β-catenin/TCF-dependent luciferase reporter, and human Frizzled-1 expression plasmids) and a control cell line (stably transfected with FOPFlash, a negative control reporter with mutated β-catenin/TCF binding elements)36 (link) were kindly provided by Prof. Sangtaek Oh, Kookmin University (Seoul, Korea). SW480 and CCD-18Co cells were cultured in RMPI. The other cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (10 μg/ml) at 37 °C.

SW480 CRCs, A549 lung cancer cells, HeLa CCL2 cervical cancer cells, and U-2 OS osteosarcoma cells were obtained from the American Type Culture Collection (Manassas, VA). CCD-18Co cells were obtained from the Korean Cell Line Bank (Seoul, Korea).The SW480, CCD-18Co and HeLa CCL2 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin, and 100 μg/ml streptomycin. A549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 μg/ml penicillin, and 100 μg/ml streptomycin. U-2 OS cells were cultured in McCoy's 5A medium supplemented with 10% FBS, 100 μg/ml penicillin, and 100 μg/ml streptomycin. The cultured cells were incubated under a humidified atmosphere of 5% CO₂ at 37 °C.