Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: Co-localization between TF and EphA2 in MDA-MB-231 cells. a EphA2 cleavage is independent of PAR2. MDA-MB-231 cells were pretreated with 50 μg/ml anti-TF antibody 10H10 or 100 μg/ml PAR2-blocking antibody for 30 min followed by 10 nM FVIIa for 6 h. Images show representative Western blots. The 10H10 antibody was verified to prevent IL8 mRNA induction by FVIIa (graph). MDA-MB-231 cells were pre-treated with 50 μg/ml 10H10 antibody for 30 min, and 10 nM FVIIa was added for 1 h. FVIIa increased IL8 mRNA to 224.6 ± 0.3 %, whereas preincubation with mab10H10 abolished induction to 112.6 ± 1.2 %. N = 2, results are presented as percent of untreated control with error bars indicating the standard deviation. b EphA2 and TF in proximity were detected in MDA-MB-231 cells using an in situ proximity ligation assay with antibodies towards TF and EphA2. Red signal corresponds to TF-EphA2 complexes, blue signal represents DAPI-stained DNA. c Confocal micrographs showing MDA-MB-231 cells stained for EphA2 (green signal) and TF (red signal). Blue signal represents DAPI-stained DNA. Scale bar represents 20 μm

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Blocking Assay, Western Blot, Control, Standard Deviation, In Situ, Proximity Ligation Assay, Staining

Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: Overexpression of TF is required for EphA2 co-localization and cleavage in U251 cells. a - b U251 cells were transfected with a plasmid encoding human TF or treated with Lipofectamine alone as control. The figure shows confocal micrographs of control ( a ) or TF transfected ( b ) U251 cells stained for EphA2 (green signal) and TF (red signal). Blue signal represents DAPI-stained DNA. Scale bar represents 20 μm. c Lipofectamine-treated or TF-transfected U251 cells were treated with 10 nM FVIIa for the indicated time points. Samples were analyzed for EphA2 and TF expression by Western blot. N = 2–3, representative blots are shown

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Staining, Expressing, Western Blot

Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: EphA2 co-localize with f-actin and FVIIa stimulation potentiates ephrin-A1-induced cell rounding and retraction fiber formation in MDA-MB-231 cells. a EphA2 co-localizes with the actin cytoskeleton in MDA-MB-231 cells. Confocal micrographs showing EphA2 (red signal) and actin fibers stained by phalloidin (green signal). Blue signal represents DAPI-stained DNA. b MDA-MB-231 cells were plated on collagen IV and treated with 10 nM FVIIa for 1 h prior to stimulation with 1 μg/ml ephrin-A1 for 10, 30 and 60 min. Cells were fixed and stained with FITC-conjugated phalloidin and micrographs generated by epifluorescence microscopy. Ephrin-A1 treatment induced cell rounding and retraction fiber formation, which was potentiated by FVIIa to 49.7 ± 4.4 % vs 29.3 ± 4.1 % at 10 min ( p =0.009), 30.9 ± 8.9 % vs 16.2 ± 1.6 % at 30 min ( p = 0.08) and 25.4 ± 7.5 % vs 13.3 ± 2.9 % at 60 min ( p = 0.010). FVIIa alone did not increase cell rounding and retraction fiber formation compared to untreated cells (7.3 ± 4.5 % vs 5.2 ± 2 %, p = 0.49). N = 3, results are expressed as means ± SD of the percentage of rounded cells with retraction fibers. Representative images are shown

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Staining, Generated, Epifluorescence Microscopy

Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: EphA2 is tyrosine phosphorylated by FVIIa and ephrin-A1. a MDA-MB-231 cells were treated with 10 nM FVIIa and samples were analyzed by Western blot for EphA2 phosphorylation on tyrosine 588 (Y588) and total EphA2 protein. FVIIa increased the phosphotyrosine signal to 110.0 ± 1.02 % ( p < 0.001) after 30 min and 129.2 ± 9.82 % ( p = 0.0067) after 60 min. N = 3, results are presented as means ± SD. b MDA-MB-231 cells were pretreated with 10 nM FVIIa for 1 h and then stimulated with 1 μg/ml ephrin-A1-Fc for 5 or 10 min. c MDA-MB-231 cells were pretreated with 10 nM FVIIa for 6 h and then stimulated with 1 μg/ml ephrin-A1-Fc for 5 min. Samples were analyzed by Western blot as in ( a ). N = 2–3, representative gels are shown

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Western Blot, Phospho-proteomics

Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: Association between TF and EPHA2 expression in colorectal cancer

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: Associations between TF and EPHA2 positive colorectal cancers and patient and tumor characteristics

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques:

Journal: BMC Cancer

Article Title: Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

doi: 10.1186/s12885-016-2375-1

Figure Lengend Snippet: EphA2 and TF are co-expressed in a colorectal cancer. Representative images of immunohistochemistry stainings for TF and EphA2. Brown color represents positive staining. a Serial sections from specimen with high expression of TF ( left panel ) and EphA2 ( right panel ). Original magnification 20×. b Serial sections from specimen with scattered positivity for TF ( left panel ) and EphA2 ( right panel ) localized to necrotic areas and budding tumor cells. Original magnification 40 ×

Article Snippet: Antibodies towards EphA2 (6997), pS897-EphA2 (6347), pY588-EphA2 (12677) and GAPDH (2118) were from Cell Signaling Technology.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 1. JESS capillary-based Westerns demonstrate canonical EphA2-pY589 activation is restricted to the lens epithelial cells while non-canonical EphA2-pS898 phosphorylation is present in epithelial cells and cortical fibers of 6-week-old, 4-

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Activation Assay, Phospho-proteomics

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 2. Pan-EphA2 is localized to epithelial and fiber cells with enriched signal in fiber cells. Longitudinal lens sections from 6-week-old, 4-month-old, and 8-month-old ephrin-A5+/+ and ephrin-A5-/- mice were stained with pan-EphA2 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). The pan-EphA2 signal was visible in epithelial cells (arrows) and was strongly localized within the cortical fiber cell region (asterisks). This pattern remained consistent between age-matched controls and ephrin-A5-/- lenses. Scale bar, 50 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Staining

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 3. In lens cells, pan-EphA2 is localized to the cell membrane. Longitudinal lens sections from 4-month-old control and ephrin- A5-/- mice were stained with pan-EphA2 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). Images of the equator region were taken in sequence along similar areas of the lens. Fluorescence signal from pan-EphA2 appeared along the cell membrane within the lens. In anterior epithelial cells, there was increased pan-EphA2 signal at the apical-apical junction (arrowheads) between epithelial cells and fiber cells. In equatorial epithelial cells, pan-EphA2 was enriched at the basal (open arrowheads) membrane between epithelial cells and the lens capsule and at the apical membrane (arrowheads). In cortical lens fiber cells, pan-EphA2 outlined the cell membrane. There are no obvious differences between control and ephrin-A5-/- sections. Scale bars, 20 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Membrane, Control, Staining, Sequencing, Fluorescence

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 4. In lens fiber cells, pan-EphA2 is localized to the cell membrane and enriched along the short sides. Lens sections in the cross orientation from 4-month-old control and ephrin-A5-/- mice were stained with pan-EphA2 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). E denotes the epithelial cells, and images of the fiber cells were taken in sequence along similar areas of the lens equator. Fluorescence from pan-EphA2 was along the cell membrane of peripheral and mature fiber cells in the lens cortex region (asterisks). The signal was enriched along the short sides of lens fiber cells. Scale bar, 20 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Membrane, Control, Staining, Sequencing, Fluorescence

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 5. Canonically activated EphA2-pY589 protein is localized exclusively in epithelial cells. Longitudinal lens sections from 6- week-old, 4-month-old, and 8-month-old ephrin-A5+/+ and ephrin-A5-/- mice were stained with EphA2-pY589 (green) antibody, phalloidin (F- actin, red), and DAPI (nuclei, blue). The EphA2-pY589 fluorescence highlighted the lens epithelial cells (arrows) across all age-matched control and ephrin-A5-/- lenses. Scale bar, 50 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Staining, Fluorescence, Control

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 6. Canonically active EphA2-pY589 protein is enriched at basal and apical membranes of lens epithelial cells. Images of the equator region were taken in sequence along similar areas of the lens. In 4-month-old control and ephrin-A5-/- longitudinal lens sections stained with EphA2-pY589 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue), the EphA2-pY589 signal was visible in anterior lens epithelial cells with enrichment at the basal (open arrowheads) and apical (arrowheads) membranes. There was increased EphA2-pY589 staining signal in equatorial epithelial cells with enrichment at the basal (open arrowheads) and apical (arrowheads) membranes. EphA2- pY589 staining was also visible between equatorial epithelial cells along the lateral membranes. There are no obvious differences in staining signal and localization between control and ephrin-A5-/- sections. Scale bar, 20 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Sequencing, Control, Staining

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 7. Non-canonically activated EphA2-pS898 protein is localized in epithelial cells and mature fiber cells. Longitudinal lens sections from 6-week-old, 4-month-old, and 8-month-old ephrin-A5+/+ and ephrin-A5-/- mice were stained with EphA2-pS898 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). Fluorescence of EphA2-pS898 was found within the epithelial cells (arrows) and as a band within mature fiber cells in the lens cortex (asterisks). The staining pattern remained consistent between age-matched control and ephrin-A5-/- lenses. There was a noticeable narrowing and intensification of the EphA2-pS898 band of signal in the cortical fiber cells region with age, which is likely due to compression of fiber cells as the lens continues to grow. Scale bar, 50 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Staining, Fluorescence, Control

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 8. Non-canonically active EphA2-pS898 protein is enriched in equatorial lens epithelial cells. Longitudinal lens sections from 4-month-old control and ephrin-A5-/- mice were stained with EphA2-pS898 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). Images of the equator region were taken in sequence along similar areas of the lens. Mature fiber cells with strong EphA2-pS898 cannot be seen in these images of the peripheral fibers. EphA2-pS898 signal was found to weakly stain anterior epithelial cells, and there was increased signal in equatorial epithelial cells (arrows). There are no obvious differences between control and ephrin-A5-/- lenses. Scale bar, 20 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Control, Staining, Sequencing

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 9. In lens fiber cells, EphA2-pS898 signal is localized to the cell membrane of mature fibers. Lens sections in the cross orientation from 4-month-old control and ephrin-A5-/- mice were stained with EphA2-pS898 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). E denotes the epithelial cells, and images of the fiber cells were taken in sequence along similar areas of the lens. EphA2- pS898 staining signals were enriched in mature lens fiber cells (asterisks), and these signals appear to be all around the cell membrane without preference between the broad and short sides of fibers. The staining signal was comparable between control and ephrin-A5-/- lenses. Scale bar, 20 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Membrane, Control, Staining, Sequencing

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 10. In lens fiber cells, EphA2-pS898 signal is localized to the cell membrane of mature fibers. Lens sections in the cross orientation from 8-month-old control and ephrin-A5-/- mice were stained with EphA2-pS898 (green) antibody, phalloidin (F-actin, red), and DAPI (nuclei, blue). EphA2-pS898 signal was present in mature fiber cells (asterisks). The signal appeared cytosolic and did not have obvious membrane localization. The staining signals were comparable between control and ephrin-A5-/- sections. Scale bar, 20 μm.

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Membrane, Control, Staining

Journal: Aging

Article Title: Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

doi: 10.18632/aging.206144

Figure Lengend Snippet: Figure 11. A summary of the localization and protein levels of canonically active EphA2-pY589 and non-canonically active EphA2-pS898 in control and ephrin-A5-/- lenses from 6-week-old, 4-month-old, and 8-month-old mice. Canonical ligand- mediated EphA2-pY589 is only found in lens epithelial cells, and protein levels do not change with age. Non-canonical ligand-independent activation of EphA2-pS898 is found in epithelial cells and mature fiber cells in the lens cortex. The levels of EphA2-pS898 increase with age, and in ephrin-A5-/- epithelial cells, there is an increase compared to the control in 8-month-old mice. Cartoon not drawn to scale. Modified from [38].

Article Snippet: Detection of protein was performed by loading lens epithelial cells (~0.4 μg/μL) and cortical fiber cells (3 μg/μL) with 1:50 of a rabbit anti-EphA2 antibody (6997, Cell Signaling Technology), 1:50 of a rabbit anti-EphA2-pS897 antibody (6347, Cell Signaling Technology) or 1:50 of a rabbit-EphA2-pY588 antibody (12677, Cell Signaling Technology).

Techniques: Control, Activation Assay, Modification