Journal: Oncology Letters

Article Title: Macrophages activate mesenchymal stem cells to acquire cancer-associated fibroblast-like features resulting in gastric epithelial cell lesions and malignant transformation in vitro

doi: 10.3892/ol.2018.9703

Figure Lengend Snippet: Gastric epithelial cells incubated with CM from macrophage-hucMSCs exhibit several properties of stem cells. (A) GES-1 cells were incubated with CM from hucMSCs or macrophage-hucMSCs for 48 h, and then seeded in non-adherent culture conditions for spheroid formation. Depicted are representative images of spheroid colonies after 15 days. Magnification, ×400; scale bar, 50 µm. (B) Quantification of the soft agar colonies shown in (A). (C) Western blot analysis of Nanog, SOX2, and BMI-1 in GES-1 cells (the controls) and GES-1 cells co-cultured with CM from hucMSCs and macrophage-hucMSCs after 48 h. The expression of all analyzed proteins is significantly enhanced in the GES-1 cells cultured with CM from macrophage-hucMSCs, compared with the control. (D) Representative image of single-colony formations of GES-1 cells incubated with CM from hucMSCs or CM from macrophage-hucMSCs. (E) The number of colonies is depicted as mean ± standard deviation. *P<0.05, # P<0.01; GES-1 cells treated with medium only served as the control (magnification, ×100). Macrophage-hucMSC, human umbilical cord-derived mesenchymal stem cells pre-cultured with macrophages for 48 h; CM, conditioned medium; SOX2, SRY-box 2; BMI-1, polycomb complex protein BMI-1.

Article Snippet: The membranes were incubated with 5% skimmed milk to block non-specific protein at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies at a dilution of 1:800 for rabbit polyclonal anti-N-cadherin (cat. no. BS2224; Bioworld Technology, Inc., St. Louis Park, MN, USA), 1:1,000 for rabbit polyclonal anti-E-cadherin (cat. no. BS1098; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-vimentin (cat. no. BS1855; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-α-SMA (cat. no. BS8796; Bioworld Technology, Inc.), 1:800 for rabbit polyclonal anti-B-cell lymphoma-2 (Bcl-2) (cat. no. BS70205; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-Bcl-2-associated X (Bax) (cat. no. BS1030; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-SOX2 (cat. no. BS6161; Bioworld Technology, Inc.), 1:1,000 for rabbit polyclonal anti-Bmi-1 (cat. no. BS6015; Bioworld Technology, Inc.), 1:10,000 for rabbit polyclonal anti-β-actin (cat. no. AP0060; Bioworld Technology, Inc.), 1:1,000 for mouse monoclonal anti-FAP (cat. no. sc-71094; Santa Cruz Biotechnology, Inc.,) and 1:1,000 for rabbit polyclonal anti-Nanog (cat. no. sc-33759; Santa Cruz Biotechnology, Inc.).

Techniques: Incubation, Western Blot, Cell Culture, Expressing, Standard Deviation, Derivative Assay