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. 2015 Mar 10;108(5):1013–1026. doi: 10.1016/j.bpj.2015.01.005

Figure 1.

Figure 1

Effect of gefitinib on EGFR homodimerization. (a) FRET/FLIM images of HCC1954 cells that were stimulated with EGF in the absence or presence of gefitinib pretreatment, and then fixed and stained with Alexa 546- and Dy Light 649-labeled antibodies (clone F4) that recognize the intracellular domain of EGFR. This corresponds to an increase in the apparent average FRET efficiency from 1.2% without gefitinib treatment (τEGF = 2.46 ± 0. 19 ns) to 7.5% with gefitinib treatment (τgefitinib+EGF = 2.30 ± 0.17 ns; p < 0.001). Fluorescence lifetimes were calculated by a monoexponential fit to the data. Scale bar is 50 μm for all images. (b) Mono- and biexponential pixel fittings of the individual FRET/FLIM images of D-only-labeled cells (Alexa 546) and DA (Alexa 546, Dy Light 649)-labeled cells before and after gefitinib treatment, respectively. (c) The partial contributions of the noninteracting, D-only-labeled species and interacting DA-labeled species were deconvoluted and histograms of the EGFR homodimers subpopulations before (green) and after (red) gefitinib treatment were plotted. Fractional-intensity histograms were accumulated over five images and normalized for direct comparison. The homodimer percentage increased from 17% to 24% after the gefitinib treatment, which corresponds to ∼40% enhanced dimerization. (d) Single-molecule signature FRET images of an individual molecule. Single-molecule images of EGF-stimulated fixed cells fluorescently labeled with the Alexa Fluor 555/Alexa Fluor 647 D38-B1 FRET pair were acquired with dual-excitation TIRF (Fig. S2 b). At t = 3.7 s, only the acceptor emission can be detected. At t = 11.4 s, the donor signal arises upon acceptor photobleaching. Scale bar is 1 μm for all images. (e) Corresponding spFRET track showing anticorrelated D and A signals (E = 98%). A similar track was recorded for a gefitinib-pretreated molecule (E = 81%). (f) GFP fluorescence and donor FLIM images (of Alexa 546-labeled anti-EGFR antibody) of fixed HCC1954 cells transfected with GFP cs DEP-1 (Alexa 546 τcsDEP-1 = 2.69 ± 0. 23 ns) and WT DEP-1 (Alexa 546 τWTDEP-1 = 2.45 ± 0. 11 ns) plasmids for inactive and overexpressed DEP-1 (EGFR) phosphatase, respectively, without EGF stimulation. The apparent average FRET efficiency increased from 3% for inactive DEP-1 phosphatase to 5% for overexpressed DEP-1 (p < 0.001). To see this figure in color, go online.