Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Inhibition of FGF signaling did not activate Src or FAK. BAEC were transduced with Ad-GFP or Ad-FGFR1DN. Cells in normal growth medium were lysed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. B , C , D , Quantitative analysis of pY416 Src (B), pY527 Src (C), pY397 Fak (D) shown in Fig. 3A. The value of NT, standardized with β-tubulin, was designated as 1. (n = 3). E , F . Reduced SHP2 expression in cells lacking FGF signaling. Western analysis using BAEC total cell lysates left untreated or transduced with either Ad-GFP or Ad-FGFR1DN. G , Quantitative analysis of SHP2 levels shown in Fig. 3F. The value of Ad-GFP at MOI 37.5, standardized with β-tubulin, was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with Ad-GFP, MOI 37.5). H , I . VE-cadherin-SHP2 interaction was disrupted by FGF signaling inhibition. BAEC were transduced with Ad-GFP or Ad-FGFR1DN. Cells were lysed and immunoprecipitated (IP) with anti-VE-cadherin (H) or anti-SHP2 (I) and subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. NT denotes no transduction. J . Catalytically inactive, dominant-negative SHP2 increased Y658 VE-cadherin phosphorylation. BAEC were transduced with lentivirus wild-typeSHP2 (WT) or dominant-negative-SHP2 (C/S). Cells were lysed and total cell lystes were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Inhibition, Transduction, SDS Page, Western Blot, Expressing, Immunoprecipitation, Dominant Negative Mutation, Phospho-proteomics

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. SHP2 mRNA levels were not decreased in endothelial cells lacking FGF signaling. Quantitative RT-PCR analysis of total RNA isolated from BAEC. Total RNA was isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN. SHP2 mRNA levels were measured with real-time PCR and normalized to GAPDH expression (Mean ± SD, * P <0.05, by t-test compared with NT). NT denotes no transduction. B . Western blotting of total cell lysates isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN and treated with 10 µg/ml cycloheximide for up to 36 hours. C . Quantitative analysis of SHP2 Western analysis described in B. The value at time point 0 was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with Ad-GFP). D . SHP2 is degraded via the lysosomal pathway in the absence of FGF signaling. Confluent BAEC transduced with either Ad-GFP or Ad-FGFR1DN were treated with 1 µM MG132, 20 µM lactacystin, 20 µM chloroquine or 25 mM NH 4 Cl for 24 hr. Total cell lysates were analyzed by Western blot. E . Quantitative analysis of SHP2 expression shown in Fig. 2D. The value of Ad-GFP control (DMSO) treatment, standardized with β-tubulin, was designated as 1. (n = 3, Mean ± SD, * P <0.05, by t-test compared with Ad-FGFR1DN control).

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Quantitative RT-PCR, Isolation, Transduction, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Increased phosphorylation of VE-cadherin Y658 in cells lacking FGF signaling was restored to the basal level by SHP2 overexpression. BAEC were transduced with Ad-FGFR1DN and Ad-GFP or Ad-SHP2. Cells were lysed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. B . Quantitative analysis of SHP2 shown in Fig. 5A. The value of NT, standardized with β-tubulin, was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with NT, ** P <0.05, by t-test compared with Ad-FGFR1DN+Ad-GFP). C . SHP2 overexpression restored p120-catenin/VE-cadherin association. BAEC were transduced with Ad-FGFR1DN and Ad-GFP or Ad-SHP2. Total cell lysates were isolated and immunoprecipitated with VE-cadherin antibody. Immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies using the same membrane after stripping and reprobing. D , E , Immunostaining of quiescent and fully confluent BAEC transduced with Ad-FGFR1DN (D), or Ad-SHP2 and Ad-FGFR1DN (E). Cells were stained for VE-cadherin (green), SHP2 (red), and HA (FGFR1DN, blue). Arrows indicate gap formations between cells. F . Quantitative analysis of immunostaining evaluating the gap formation. Percent of gap area in each image was calculated using NIH Image J software using 6 different images. Data shown as mean ± SD *: P <0.05 by t-test. Scale Bars: 10 µm. G . SHP2 overexpression rescued the FGFR1DN effect on endothelial permeability. BAEC were transduced with Ad-SHP2 and Ad-FGFR1DN, and endothelial monolayer permeability was evaluated with the ECIS system. Transendothelial resistance was measured every 5 minutes for 17 hours after the onset of adenoviral transduction. NT denotes no transduction.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Phospho-proteomics, Over Expression, Transduction, SDS Page, Western Blot, Isolation, Immunoprecipitation, Membrane, Stripping Membranes, Immunostaining, Staining, Software, Permeability

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Under normal circumstances, SHP2 is associated with VE-cadherin and dephosphorylates Y658 site of VE-cadherin, which results in p120-catenin coupling, enhancing VE-cadherin retention at adherens junctions. FGF signaling is required for VE-cadherin-SHP2 interaction. B . In cells expressing the FGFR1DN construct, SHP2 is downregulated and is not able to associate with VE-cadherin due to the lack of FGF signaling, which increases phosphorylation level of VE-cadherin Y658, leading to decoupling of p120-catenin; therefore, VE-cadherin stability at cell-cell junctions is impaired.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Expressing, Construct, Phospho-proteomics