Fig. 3

Adipocytes overexpressing Grem2 inhibit the proliferation and invasion of breast cancer cells. A–C After differentiation of 3T3-L1-mock or 3T3-L1-Grem2 pre-adipocytes for 9 days, each cell line was mixed with MTV/TM-011 cells and seeded into ultra-low attachment 96 wells. Before mixing the cells, adipocytes were fluorescently stained green and breast cancer cells red (A). The growth of spheroids by 3D co-culture was observed under a fluorescence microscope (B) and their proliferation level was measured using the CellTiter-Glo® 3D Cell Viability Assay (C). Scale bar = 100 µm. Two-sided t-test. ***, p < 0.001. D–F After differentiation of 3T3-L1-mock or 3T3-L1-Grem2 pre-adipocytes for 9 days, each cell line was mixed with MTV/TM-011 cells and performed 3D invasion assay (D, E). The invasion area was measured using the Image J (F). Scale bar = 100 µm. Two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant