Journal: PLoS ONE

Article Title: Phosphorylation of Akt and ERK1/2 Is Required for VEGF-A/VEGFR2-Induced Proliferation and Migration of Lymphatic Endothelium

doi: 10.1371/journal.pone.0028947

Figure Lengend Snippet: A–C : PROX1 ( A ) is localized in the nucleus ( B ) in primary human LECs ( C ). D : Cell viability/proliferation was measured with Cell Titer Blue reagent after culturing LECs for 48 hours in EGM-2MV media (positive control), reduced-serum media (negative control), or with VEGF-A (100 ng/ml) in the presence or absence of r84 (500 molar excess), a functional blocking antibody against VEGFR1 (500 molar excess), or control IgG (500 molar excess). r84 blocked VEGF-A-induced proliferation/viability of LECs whereas the other antibodies had no effect. E : LECs were seeded in the upper chamber of a transwell insert and allowed to migrate overnight toward EGM-2MV (positive control), reduced-serum media (negative control), or VEGF-A (100 ng/ml) in the presence or absence of r84 (500 molar excess), a functional blocking antibody against VEGFR1 (500 molar excess), or control IgG (500 molar excess). The number of LECs that migrated to the lower chamber was counted and normalized to the positive control. r84 blocked VEGF-A-induced migration whereas the other antibodies had no effect. For panels C and D, significance tested by ANOVA. Asterisk P<0.05 compared to VEGF-A. ns = not significant compared to VEGF-A.

Article Snippet: The VEGFR1 functional blocking antibody (6.12) was from ImClone.

Techniques: Positive Control, Negative Control, Functional Assay, Blocking Assay, Control, Migration

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Decreased αSMA promoter activity and αSMA protein expression in bleomycin-injured mice treated with IGF-1 receptor blocking antibody (A12). (A) Representative H&E sections of αSMA-GFP mice at day 21 after bleomycin injury. Fibrotic regions of lung parenchyma are indicated by ( ) and normal lung parenchyma are indicated by ( n ). (B) αSMA immunostaining of bleomycin-injured lung in an αSMA-GFP mouse. Note the overlap of αSMA staining (red) with αSMA-GFP expression (green) in the peribronchiolar fibrotic region indicated by ( ). Scale bars represent 100 μm. (C) (Left) Representative fluorescent images of αSMA-GFP mice treated with A12 (b and d) showed less αSMA promoter activity as indicated by GFP (green) positive cells, compared to control mice (a and c) at d21 after bleomycin instillation. (Right) Percentage of αSMA-GFP + cells/total number of DAPI + cells, quantification by NIH ImageJ (n = 4 mice/group, mean ± SEM). (D) (Left) Representative images of αSMA staining by immunofluorescent microscopy of the same A12-treated mice (b and d) compared to control mice (a and c) at d21 after bleomycin instillation. Large airways and vasculature staining for αSMA, indicated by an asterisk (*), were masked in the analysis. Interstitial staining, indicated by an arrow ( ), was included in the analysis. (Right) Ratio of αSMA staining area per DAPI + area (n = 4 mice/group, mean ± SEM).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Activity Assay, Expressing, Blocking Assay, Immunostaining, Staining, Control, Microscopy

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Effect of matrix stiffness on response to IGF-1 treatment. (A) MLF on tissue culture plate or collagen I-coated tissue culture plate (stiff substrates) were treated with IGF-1 (100 ng/ml), TGFβ (1 ng/ml) or IGF/TGFβ (100 ng/ml and 1 ng/ml, respectively), or serum-free media (negative control) for 24 hr. (B) MLF on collagen I (1 mg/ml) hydrogel (soft substrate) were treated with IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) with A12 (40 μg/ml) or PI3 kinase inhibitor LY294002 (Ly, 50 μM;) for 24 h. (C) MLF isolated from bleomycin-injured C57Bl6 mice were treated with the indicated cytokine. Real time PCR analyses of myofibroblast markers Acta2 , Col1a1 , and Col3a1 were performed. Data were normalized to HPRT expression. Y-axis represents fold increase compared to serum-free control (n = 3, mean ± SEM, * p < 0.05 compared to serum-free control).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Negative Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: IGF-1 treatment increases αSMA stress fibers. A . MLF treated with IGF-1 (100 ng/ml), TGF-β1 (10 ng/ml) or IGF-1/TGF-β1 (100 ng/ml and 10 ng/ml, respectively) with or without A12 (40 μg/ml) for 24 hr. Negative control is serum free media. Cells were co-stained for F-actin (red) and αSMA (green). B . The mean ratio (±SEM) of αSMA stress fiber (+) fibroblasts over all F-actin (+) fibroblasts is presented in the bar graph. * p < 0.05 compared to serum-free control. ** p < <0.01 compared to serum-free control.

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Negative Control, Staining, Control