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Fig. 4. PKC
and Rac activities regulate the interaction of fascin and PKC in migrating cells. (A,B) SW480 IKD-F11 Fas–cells transiently transfected with GFP-Xtfascin alone (bottom line in A) or GFP-Xtfascin and mRFP-PKC (all other panels and in B) were plated on 15 nM LN for 2 hours, with or without pretreatment with 1 µM BIM for 30 minutes or 100 µM NSC23766 for 4 hours, then fixed, mounted and imaged using FLIM to measure FRET. (A) Intensity multiphoton GFP images from each cell (donor) and, where relevant, an epifluorescence image for mRFP (acceptor). Lifetime images are depicted by a pseudocolour scale with red as low lifetime (1.7 nseconds) and blue as high (2.3 nseconds). (B) Histogram demonstrating mean cumulative FRET efficiency data from 16 cells per condition and three independent experiments. The graph represents the range of FRET efficiencies seen per cell in each condition, normalised to pixel intensity, and demonstrates a clear leftward shift in BIM- and NSC-treated samples where FRET is inhibited. (C) A kinase-dead form of PKC, PKCK380A, does not interact with fascin in cells migrating on laminin. The cumulative FRET efficiency graph shows the mean FRET efficiency for wild-type and kinase-dead PKC. Each column represents the mean from seven to nine cells per condition and three independent experiments. Insets show the GFP and RFP intensities, and lifetime plot from a representative cell expressing PKC–K380A. Pseudocolour scale as in A.
