Figure 3.

Single-molecule analysis of the effect of the TKI gefitinib on the EGFR homodimer stability. (a and b) Histograms of the duration of dimerization events fitted to a single exponential with k_off^EGF = 1.10 ± 0.07 s⁻¹ and K_off^gefit+EGF = 0.87 ± 0.09 s⁻¹ when cells were pretreated with gefitinib. (c and d) Ensemble correlated motion analysis of two-color single-particle tracking data for EGF-stimulated cells in the absence and presence of gefitinib pretreatment, respectively, for all two-color tracked molecules. The average receptor displacement (red) and the degree of uncorrelated motion (blue) are plotted as function of the separation distance. Correlated motion at small separation distances indicates dimerization. Increased correlation at short distances can be seen by the decrease in the uncorrelated jump distance (blue) when the two receptors approach one another. The diffusion behavior was monitored via the jump magnitude (red). Decreased mobility with dimerization (i.e., a drop in jump magnitude at short distances) was observed independently of gefitinib treatment. (e) Dissociation rates calculated from five individual experiments show a consistent reduction in k_off (p < 0.05) with TKI pretreatment independently of the type of TKI used (lapatinib, gefitinib, or AG 1478). (f) Decreased sample heterogeneity after gefitinib pretreatment. The fraction of colocalized molecules shifted from 3.59% ± 1.56% to 1.95% ± 0.63% after gefitinib pretreatment. To see this figure in color, go online.