Figure 5

Tetracycline-induced cell fusion in p14 -engineered T-REx-293 cells. (a) Bright-field microscopy, DAPI staining (white or magenta) and phalloidin-FITC (green) of THFU-4 cells (a representative clone of T-REx-293 cells carrying the fusion module), treated or not with tetracycline for 24 h. Untreated cells (left column) grow normally, without obvious signs of fusion, nuclei remaining separate and surrounded individually by cell membranes, shown by phalloidin stain of cortical actin filaments in the bottom row. Cells in which the fusion construct is induced show formation of large, multinucleate cells in bright field illumination (top row), formation of ‘rosettes’ of tightly apposed nuclei (middle row), with cortical actin (green) surrounding the whole groups of nuclei rather than each individual one (bottom row). (b) Cells grown in calcium-free medium showed fewer fusion events, as expected (33). (c) Evidence of fusion between tetracycline-induced cells from clone THFU-10 (another representative clone) and MDCK cells transiently expressing mCherry: unfused MDCK cells appear as small, deep red individual cells (white arrow). The red is also seen (black arrows) in large syncytia, implying that the THFU-10 cells can fuse with cells not themselves containing the fusion module. Scale bars: 100 μm.