Journal: bioRxiv

Article Title: Interdependence of EGFR with PTPs on juxtaposed membranes generates a growth factor sensing and responding network

doi: 10.1101/309781

Figure Lengend Snippet: (A) PTP X and EGFR mRNA expression in MCF7 cells measured by microarray analysis (relative to GAPDH mRNA). AS: anti-sense. (B) Estimating EGFR-mTFP occupancy with EGF-Alexa647 in live cells: EGF-Alexa647/EGFR-mTFP quantified in single cells (data points) upon increasing EGF-Alexa647 doses was fitted (line) with a receptor binding kinetics model (STAR Methods). (C) Fraction of phosphorylated receptor, quantified by PTB-mCherry translocation to the PM ( star Methods) for different EGF-Alexa647 doses (single cell profiles). (D) Single cell EGFR-mTFP phosphorylation dose response profiles (average shown in ). The estimated fractions of phosphorylated vs. liganded EGFR-mTFP are plotted and color-coded by the average EGFR-mTFP fluorescence intensity per cell. (E) Quantification of PTB-mCherry translocation kinetics to PM-localized EGFR-mTFP. MCF7 cells were stimulated with a saturating EGF-Alexa647 dose (320ng/ml) and successive images were acquired every 20s (n=10). Translocated PM fraction of PTB-mCherry ( ) converged to a steady state level in ~1.5min, which was within the time frame of successive EGF-Alexa647 dose administration . (F) Estimation of EGFR-QG-mCitrine occupancy with EGF-Alexa647 in live cells (n=30, N=3) from fluorescence anisotropy microscopy is equivalent to the corresponding estimation from confocal microscopy with EGFR-mTFP . (G) Dependency of the EGFR dimerization state with increasing EGF-Alexa647 doses determined by fluorescence anisotropy microscopy (n=30, N=3). (H) Dimensionality reduction from Cartesian (x, y) to normalized radial (r) distribution of quantity (Q) between the plasma (PM) and the nuclear (NM) membrane. (I) Average spatial-temporal maps (STMs) of EGFR-mCitrine intensity obtained from live cells stimulated with 200ng/ml S-EGF (n=16, N=3; top) and 5P-EGF (n=14, N=2; bottom). (J) Corresponding STMs of EGF-Alexa647 fluorescence. (K) Quantification of recycling dynamics of ligandless EGFR-mCitrine upon 200ng/ml 5P-EGF. Top: PM fraction of ligandless EGFR-mCitrine in single cells. Model-based estimation of the steady state level (95% confidence bounds; see STAR Methods) is shown with black line (inside red dashed lines). Middle: compartment-model-based fitting on 4-28min interval for the cells shown in the bottom panel of (estimated rates: k in =0.125, k rec =0.046, STAR Methods). Bottom: Linear dependency between (k in , k rec ) reflects that similar PM steady state levels of ligandless EGFR are maintained by recycling in single cells (x: average (k in , k rec ); STAR Methods).

Article Snippet: Primary antibodies: Mouse monoclonal antibody PY72 ( ) (InVivo Biotech Services, Henningsdorf, Germany), rabbit anti EGFR pY 1068 (Cell Signaling; 1:400), goat anti EGFR (R&D Systems; 1:300).

Techniques: Expressing, Microarray, Binding Assay, Translocation Assay, Fluorescence, Microscopy, Confocal Microscopy, Membrane

Journal: bioRxiv

Article Title: Interdependence of EGFR with PTPs on juxtaposed membranes generates a growth factor sensing and responding network

doi: 10.1101/309781

Figure Lengend Snippet: (A) Quantifying ectopic EGFR-mTFP expression in MCF7 cells. Average EGF-Alexa647 vs. EGFR-mTFP fluorescence in single MCF7 (green) or MCF10A cells without EGFR-mTFP (black). Histograms reflect that levels of EGF-Alexa647 binding to MCF7 with ectopic EGFR-mTFP expression (green) and MCF10A with endogenous EGFR (black) are similar. (B) EGFR Y 1068 phosphorylation (left) and Akt phosphorylation (right) in MCF7 cells ectopically expressing EGFR-mTFP (solid lines) and endogenous EGFR in MCF10A cells (dashed lines), following 5min pulsed (5P-EGF, 200ng/ml, blue) or sustained EGF stimulation (S-EGF, 200ng/ml, red), as determined by in-cell Western assay (N=3). Data are normalized to the maximum response in each respective condition (means ± SEM, N=3). (C) Representative fluorescence image series of EGF-Alexa647, EGFR-mTFP, PTB–mCherry and PTB-mCherry(magenta)/EGFR-mTFP(green) overlay from singlecell dose-response experiment. Cells were stimulated every ~1.5min with increasing EGF-Alexa647 doses (2.5-600ng/ml). Scale bar: 20μm. ( D ) Fraction of phosphorylated vs. ligand-bound EGFR-mTFP (n=21, N=2; ). Dashed lines: moving averages from single-cells; shaded bounds: standard deviations; dash-dotted lines: estimated contribution of ligandless to the fraction of phosphorylated EGFR. ( E ) Livecell fluorescence anisotropy microscopy measurements of EGFR-QG-mCitrine dimerization state as a function of the fraction of ligand-bound receptor (mean ± SEM, n=30, N=3, ). (F-H) Average spatial-temporal maps (STMs) of the estimated fraction of ligand-bound EGFR ( (F), EGF-Alexa647/EGFR-mCitrine), ligandless EGFR ( (G), 1-[EGF-Alexa647/EGFR-mCitrine]) and the fraction of phosphorylated EGFR-mTFP estimated by PTB-mCherry translocation ( (H) , PTB-mCherry/EGFR-mCitrine). Data was acquired at 1min-intervals in live MCF7 cells following 200ng/ml S-EGF (top, n=16, N=3; ) or 5P-EGF (n=14, N=2; ) stimulation. White dotted lines: trajectories representing the change in distribution of ligand-bound (F) and ligandless (G) EGFR. (I) The respective plasma membrane fractions of ligand-bound (EGF-Alexa647/EGFR-mCitrine, red) and phosphorylated EGFR (PTB-mCherry/EGFR-mCitrine, blue) derived from (F) and (H) (median ± AMD). Extracellular EGF-Alexa647 are shown in grey. (J) Dimerization state (black) and the fraction of ligand-bound EGFR-QG-mCitrine (red) at the PM for live cells following 200ng/ml S-EGF following 200ng/ml S-EGF (top, n=5, N=3) or 5P-EGF (bottom, n=5, N=3) stimulation (means ± SEM). (K) The dose-response of EGFR-mTFP phosphorylation (red, control) is significantly altered upon ectopic Rab11a S25N expression (green, p =0.02, n=12, N=3). Lines same as in (D) . (L) Scheme of EGFR trafficking dynamics: ligandless EGFR recycles via early (EE) and recycling endosomes (RE) to the PM (red arrows) whereas upon EGF binding (thin green arrow), ubiquitinated EGF-EGFRub unidirectionally traffickes via the early-to the late endosomes (LE, green arrow) to be degraded (Ø). Causal links are denoted with solid black lines.

Article Snippet: Primary antibodies: Mouse monoclonal antibody PY72 ( ) (InVivo Biotech Services, Henningsdorf, Germany), rabbit anti EGFR pY 1068 (Cell Signaling; 1:400), goat anti EGFR (R&D Systems; 1:300).

Techniques: Expressing, Fluorescence, Binding Assay, In-Cell ELISA, Microscopy, Translocation Assay, Membrane, Derivative Assay

Journal: bioRxiv

Article Title: Interdependence of EGFR with PTPs on juxtaposed membranes generates a growth factor sensing and responding network

doi: 10.1101/309781

Figure Lengend Snippet: (A) Representative images of EGFR-mTFP, EGF-Alexa647, anti-pY 1068 -Alexa568 fluorescence and overlay of pY 1068 (yellow) and EGFR-mTFP (red) prior to and after 5, 30 and 120min of 5P-EGF stimulation (200ng/ml) of MCF7 cells. Scale bar: 50μm. (B) Binding of pY 1068 -Alexa568 antibody to Y 1068 on EGFR-mTFP and EGFR Y1068F -mTFP reflects its specificity for the corresponding tyrosine phosphorylation site (mean±SD, n~100, N=1). (C) mRNA expression fold change of PTPN2, PTPRG, PTPRJ, PTPRA and DUSP3 in MCF7 cells after 24h transfection with 50nM respective siRNA. The values are relative to mRNA levels of the respective gene in cells treated with 50nM nontargeting siRNA for 24hr (N=2, and for DUSP3 N=1). (D) Left: Example of pY 1068 -Alexa568/EGFR-mTFP vs. PTPN2-mCitrine fluorescence intensity scatter plots used to determine the PTPN2-mCitrine fluorescence intensity threshold at which saturation of EGFR dephosphorylation occurs. Blue/red circles represent single cells below and above the PTPN2-mCitrine fluorescence intensity threshold, respectively; solid lines and shaded bounds: corresponding moving averages and standard deviation. The data was fitted with a function depicting the dependency of pY 1068 /EGFR-mTFP on PTP X -mCitrine intensity (steady state EGFRp assumption, STAR Methods). Right: STMs of pY 1068 /EGFR-mTFP averaged from cells below (blue box) and above (red box) PTPN2-mCitrine fluorescence intensity threshold. LUT: look-up table. (E) Effect of PTPRG-mCitrine expression (left) on STMs of EGFR-mTFP fluorescence (middle) and phosphorylation fold-change (1/PFC pY1068 -cDNA, right) reflecting the relative PTPRG-mCitrine reactivity towards pY 1068 for cells stimulated with 200ng/ml S-EGF (n~30). LUT: look-up table. (F) Columns 1-3: effect of PTPRA-mCitrine expression (Column 1) on STMs of EGFR-mTFP localization (Column 2) and phosphorylation fold-change (1/PFC pY1068 -cDNA, Column 3), which reflects the relative PTPRA-mCitrine reactivity towards pY 1068 (n~60, N=3). Column 4: effect of siRNA-mediated PTPRA knockdown on EGFR-mTFP phosphorylation fold change (PFC pY1068 -siRNA, n~45, N=3). Column 5: STM of fraction of EGFR-mTFP interacting with PTPRA C442S -mCitrine trapping mutant (α ™ , n=15-30). LUT: look-up table. (G) Effect of siRNA-mediated DUSP3 knockdown on EGFR-mTFP phosphorylation fold change (PFC pY1068 -siRNA, n~40, N=3). In (F-G), cells were stimulated with 200ng/ml 5P-EGF; transparent areas in (E-G): non-significant PFCs, p >0.05. LUT: look-up table. (H) Identifying and characterizing the interaction between EGFR and PTPRG/J-mCitrine by co-immunoprecipitation. Co-immunoprecipitated EGFR (second row) following PTPRG- (left) or PTPRJ-mCitrine (right) pull down from MCF7 cells prior to and after treatment with DPI (20min, 10μM), EGF-Alexa647 (10 min, 200ng/ml), DPI pretreatment (20min, 10μM) followed by EGF-Alexa647 (10 min, 200ng/ml) and H 2 O 2 (10 min; 2, 4 or 8 mM) by western blotting. IP input: total expressed protein, IP-PTPRG/J: PTPRG/J-mCitrine immunoprecipitated by anti-GFP antibody.

Article Snippet: Primary antibodies: Mouse monoclonal antibody PY72 ( ) (InVivo Biotech Services, Henningsdorf, Germany), rabbit anti EGFR pY 1068 (Cell Signaling; 1:400), goat anti EGFR (R&D Systems; 1:300).

Techniques: Fluorescence, Binding Assay, Expressing, Transfection, De-Phosphorylation Assay, Standard Deviation, Mutagenesis, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Interdependence of EGFR with PTPs on juxtaposed membranes generates a growth factor sensing and responding network

doi: 10.1101/309781

Figure Lengend Snippet: (A) Spatial-temporal maps (STMs) depicting EGFR-mTFP fluorescence (left) and pY 1068 phosphorylation (middle) in control cells (n~90 cells per time point, 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/PFC pY1068 -cDNA, Column 3), which reflects the relative PTPN2-mCitrine reactivity towards pY 1068 (n~60, N=3). Column 4: effect of siRNA-mediated PTPN2 knockdown on EGFR-mTFP phosphorylation fold change (PFC pY1068 -siRNA, n~45, N=3). Column 5: STM of fraction of EGFR-mTFP interacting with PTPN2 C216S -mCitrine trapping mutant (α ™ , n=15-30). (C) STMs of the same quantities as in (B) upon PTPRG-mCitrine expression (n~60, N=3; α ™ PTPRG C1060S -mCitrine n=15-30). (D) STMs of the same quantities as in (B) upon PTPRJ-mCitrine expression (n~40, N=2; α ™ PTPRJ D1205A -mCitrine, n~30). In (A-D) , cells were stimulated with 200ng/ml 5P-EGF; transparent areas: non-significant PFCs, p >0.05. (E) Effect of siRNA-mediated knockdown of PTPRG, PTPN2 and PTPRJ on the fraction of phosphorylated EGFR (a) in single MCF7 cells expressing EGFR-mCitrine (donor) and PTB-mCherry (acceptor). FLIM measurements were made prior to (grey) and 2min following saturating 320ng/ml EGF-Alexa647 stimulation (blue). amean ± SD for Control: n=14 (grey), n=17 (blue); PTPRG: n=15 (grey), n=11 (blue); PTPN2: n=9 (grey), n=8 (blue); PTPRJ: n=6 (grey), n=6 (blue); is shown. N=1-2. ** p=0.0018 and *** p<0.001. (F) Time-lapse measurements of the fraction of phosphorylated EGFR (as above) in single MCF7 cells prior to and every 5 minutes 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). Scale bar: 10 μm.

Article Snippet: Primary antibodies: Mouse monoclonal antibody PY72 ( ) (InVivo Biotech Services, Henningsdorf, Germany), rabbit anti EGFR pY 1068 (Cell Signaling; 1:400), goat anti EGFR (R&D Systems; 1:300).

Techniques: Fluorescence, Transfection, Expressing, Mutagenesis

Journal: Oncogene

Article Title: CIP4 Promotes Lung Adenocarcinoma Metastasis and Is Associated with Poor Prognosis

doi: 10.1038/onc.2014.280

Figure Lengend Snippet: CIP4 modulates EGFR signaling in NSCLC cells. (a) Serum starved H1299 V and KD1 cells were treated with EGF (100 ng/ml) for 0–20 min. Lysates were subjected to IB with the antibodies indicated on the right. Densitometry was performed and relative phospho-EGFR (pY1068) levels are shown below. Positions of molecular mass markers are shown on the left. (b) Serum starved H1299 V and KD1 cells were treated with EGF (100 ng/ml) for 0–120 min. Lysates were subjected to IB with the antibodies indicated on the right. Densitometry was performed and relative phospho-Erk levels are shown in graph below (mean ± SEM; pooled data from 2 independent experiments; * indicates a significant difference between cell lines ( P <0.05)). Positions of molecular mass markers are shown on the left.

Article Snippet: Commercial antibodies included: CIP4, N-WASP, pS2448-mTOR, pS473-Akt, pT308-Akt and Akt1/2 were from Cell Signaling Technology; EGFR, pY1068-EGFR and PE-conjugated mouse anti-human EGF receptor were from BD Bioscience; ERK1, pERK, β-actin, MMP-2, α-actinin, and EGFR were from Santa Cruz Biotechnology.

Techniques: