Fig. 4

Secreted FAF1 is transmitted to neighboring cells. a Donor cells were transfected with GFP-vector or GFP-FAF1 plasmid. At 24 h after transfection, the cultured medium was replaced with serum-free medium containing DMSO or GW4869 (10 μM) and the cells were cultured for 24 h. FAF1 was immunodepleted from CM with anti-FAF1 monoclonal antibody (1 μg/ml). Concentrated CM was applied to recipient cells for 24 h. After the CM treatment, the nuclei of the recipient cells were stained using DAPI and analyzed by confocal microscopy. b Donor cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. Concentrated CM was applied to recipient cells for 24 h. After CM treatment, CLs were analyzed by western blotting with the indicated antibodies. c Donor cells (insert) transfected with GFP-vector or GFP-FAF1 were coincubated with nontransfected recipient cells (lower compartment) in serum-free medium containing DMSO, GW4869 (10 μM), or anti-FAF1 monoclonal antibody (1 μg/ml) for 24 h. Then, the nuclei of the recipient cells were stained using DAPI and analyzed by confocal microscopy