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. 2018 Sep 26;7(3):295–309.e11. doi: 10.1016/j.cels.2018.06.006

Figure 3.

Figure 3

Spatial-Temporal Regulation of EGFR Phosphorylation by PTPN2 and PTPRG/J

(A) Spatial-temporal maps (STMs) depicting EGFR-mTFP fluorescence (left) and pY1068 phosphorylation (middle) in control cells (n ∼ 90 cells per time point for a total of ∼360 cells, N = 6 experiments) and following transfection with non-targeting siRNA pool (right, n ∼ 60, N = 4).

(B) Columns 1–3: effect of PTPN2-mCitrine expression (column 1) on STMs of EGFR-mTFP localization (column 2) and phosphorylation fold change (1/PFCpY1068-cDNA, column 3) (n ∼ 60, N = 3). Column 4: effect of siRNA-mediated PTPN2 knockdown on EGFR-mTFP phosphorylation fold change (PFCpY1068-siRNA, n ∼ 45, N = 3). Column 5: STM of fraction of EGFR-mTFP interacting with PTPN2C216S-mCitrine trapping mutant as determined by FLIM (αTM, n = 15–30, N = 2).

(C) STMs of the same quantities as in (B) upon PTPRG-mCitrine expression/siRNA-mediated knockdown (n ∼ 60, N = 3; αTM PTPRGC1060S-mCitrine n = 15–30, N = 2).

(D) STMs of the same quantities as in (B) upon PTPRJ-mCitrine expression/siRNA-mediated knockdown (n ∼ 40, N = 2; αTM PTPRJD1205A-mCitrine, n ∼ 30, N = 2). In (A) to (D), cells were stimulated with 200 ng/mL 5P-EGF; transparent areas denote non-significant PFCs, p > 0.05.

(E) Effect of siRNA-mediated knockdown of PTPRG, PTPN2, and PTPRJ on the fraction of phosphorylated EGFR (α) in single MCF7 cells expressing EGFR-mCitrine (donor) and PTB-mCherry (acceptor). FLIM measurements were made prior to (gray) and 2 min after saturating 320 ng/mL EGF-Alexa647 stimulation (blue). αmean ± SD for control: n = 14 (gray), n = 17 (blue); PTPRG: n = 15 (gray), n = 11 (blue); PTPN2: n = 9 (gray), n = 8 (blue); PTPRJ: n = 6 (gray), n = 6 (blue). N = 1–2. ∗∗p = 0.0018 and ∗∗∗p < 0.001; ns, not significant.

(F) Time-lapse measurements of the fraction of phosphorylated EGFR (as above) in single MCF7 cells prior to and every 5 min after 200 ng/mL 5P-EGF stimulation for a total of 30 min. Representative α images (left) and corresponding quantifications (right) for control (n = 4), PTPN2 (n = 5), PTPRG (n = 5), and PTPRJ (n = 4) knockdowns (N = 3, means ± SD). Scale bars, 10 μm.