Fig. 2

Osteoblasts induced PCa cell dormancy via physical contacts. (A). The schematic diagram of the co-culture model. Relative cell confluence of GFP-labelled C4-2B cells. GFP-positive areas of each time point were normalized to the ones at the co-culture seeding. (C). Cell division of C4-2B cells cultured alone or co-cultured with MC3T3-E1 cells. C4-2B cells were stained with CellTrace™ Violet (CT Violet) before seeding. FACS analyses of the CT Violet signal intensities were conducted 72 h. (D). Cell cycle analyses with PIP-FUCCI reporter. Cells with only green color, G1; Cells in red, late S/G2/M. Representative fluorescent and bright field pictures were shown. (E). C4-2B cells cultured alone or co-cultured with MC3T3-E1 for 72 h or treated with H₂O₂ for 2 h (positive control for apoptosis) were stained with Hoechst 33342 for examination of apoptotic cells (red arrows) based on the nuclear morphology. (F/H). C4-2B cells were cultured alone or co-cultured with MC3T3-E1 for 72 h. The mRNA expressions of marker genes p21 (F), NR2F1, Ki67, and Cyclin D1 (H) were examined using qRT-PCR. Human-specific primers were used, and relative expressions were calculated and plotted after normalization with GAPDH expressions. (G). C4-2B cells were treated with different conditioned media (CM) as indicated for 72 h. (I). Separation of C4-2B cells (GFP labeled) from co-culture of MC3T3-E1 cells (not labeled) was confirmed using both fluorescence microscopy (left) and species-specific PCR (right). (J). Immunoblotting of NR2F1 in C4-2B cultured alone or separated from the co-cultures. (K). The mRNA expression changes of marker genes in the 3D co-culture model. All the experiments were independently repeated at least twice. The data in curves and bar plots are presented as the mean ± SD of each set of triplicate samples. Scale bars, 50 μm (D&E), 200 μm (I). * p < 0.05, ** p < 0.01, *** p < 0.001