Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell–cell fusion

RAW264.7 cells expressing the reporter construct RFP-2xPLCδ1PH-IRES-GFP-AktPH (in red and green) in the presence of Dox (1 µg/ml) were transfected on two consecutive days with Tks5 siRNA and then stimulated with RANKL (10 ng/ml) for 48 h. Cells were exposed to DAPI at 1 µg/ml (blue) for 1 h before observation to visualize nuclei. Images were analyzed by time-lapse confocal microscopy using a laser-scanning confocal microscope (Fluoview FV10i; Olympus). Frames were taken every 3 min. Each time point is a series of 4 focal planes separated by 1 µm for each color.