Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 5

Figure 5. EGFR_T693 Phosphorylation Regulates FGFR2b Recycling

A. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.

B. Red and green pixels overlap fraction (above, left) representing the co-localization of FGFR2 with EGFR; red and far red pixels overlap fraction (above, right) representing the co-localization of FGFR2 with the recycling marker Tf; far red and green pixels overlap fraction (below, left) representing the co-localization of EGFR with the recycling marker Tf; green, red and far red pixels overlap fraction (below, right) representing the co-localization of FGFR2, EGFR, and the recycling marker Tf in T47D stimulated for 40 min. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in A and Appendix Figure S6A. *, p value<0.005 (Student's t-test).

C. Co-localization of EGFR (green), T693 phosphorylated EGFR (blue), and the recycling marker Tf (red) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.

D. Red and green pixels overlap fraction (above) representing the co-localization of EGFR with the recycling marker Tf; green, red and far red pixels overlap fraction (below) representing the co-localization of EGFR, phosphorylated EGFR, and the recycling marker Tf in T47D stimulated for 40 min. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in C and Appendix Figure S6C. *, p value<0.005 (Student's t-test).

E. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods. Scale bar, 5 μm.

F. Red and green pixels overlap fraction representing the co-localization of FGFR2 with EGFR; far red and green pixels overlap fraction representing the co-localization of EGFR with the recycling marker Tf; red and far red pixels overlap fraction representing the co-localization of FGFR2 with the recycling marker Tf; green, red and far red pixels overlap fraction representing the co-localization of FGFR2, EGFR, and the recycling marker Tf in cells stimulated for 20, 40, or 60 min. with FGF10 or TGFα. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in E. *, p value<0.005 (Student's t-test).

G. Lysates from T47D depleted of EGFR by siRNA followed by transfection with siRNA resistant wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods were used for immunoprecipitation of FGFR2 and then immunoblotted with the indicated antibodies. The inputs are shown in Appendix Figure S6F.

H. The presence (total), internalization (internalized), and recycling (cell surface) of FGFR2 in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated with FGF10 for different time periods were quantified as described (Francavilla et al., 2016) and in the section 'Quantification of the Recycling Assay". Briefly, we assessed approximately 100 cells per condition and expressed the results as the percentage of receptor-positive cells over total cells (corresponding to DAPI-stained nuclei) and referred to the values obtained at time zero. Values represent the median ± SD of N=3. *, p value<0.005 (Student's t-test).