Fig. 3. Fascin and PKC interact in living carcinoma cells migrating on LN. (A) Level of expression of GFP-Xtfascin in SW480 IKD-F11 Fas–cells compared with the endogenous fascin of SW480 cells. Cells were stained with antibody reactive with human and Xenopus fascin 1 and the mean fluorescence intensity calculated from 10 cells in each population and from three independent experiments. (B) IKD-F11 Fas–cells transiently expressing GFP-Xtfascin alone (top panels) or GFP-Xtfascin and PKC-mRFP (bottom panels) were plated on 15 nM LN for 2 hours. Transfected migrating cells were imaged over time, using FLIM to measure FRET. Panels show intensity multiphoton GFP images from each cell (donor) and, where relevant, an epifluorescence image for mRFP (acceptor) at selected timepoints. Lifetime images are depicted by a pseudocolour scale with red as low lifetime (1.7 nseconds) and blue as high (2.3 nseconds). Arrows indicate examples of strong interaction in filopodia. Representative of a total of nine experiments for control and test cells.