Journal: Theranostics

Article Title: Integrin α v β 3 -Targeted Radiotracer 99m Tc-3P-RGD 2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α v β 3 RGD 2 Therapy

doi: 10.7150/thno.6989

Figure Lengend Snippet: A : The 3D (upper panel) and transverse (lower panel) views of SPECT/CT images of a mouse with bearing MDA-MB-435 breast cancer xenografts at 5, 7, 14, 21, 28 and 35 days after inoculation of MDA-MB-435 human breast cancer cells. The animal was administered with 37 - 55.5 MBq of 99m Tc-3P-RGD 2 via the lateral tail vein. B : Microscopic images (Original magnification: ×200) of tumor slice selected from the necrotic and viable regions after immunohistochemical staining for integrin β 3 and CD31. The tumor tissue was harvested on day 28 days after inoculation of MDA-MB-435 cells. CD31 was used as a biomarker for endothelial cells on blood vessels (both mature vasculature and neovasculture). Integrin β 3 was visualized with Cy3 (red) and CD31 was visualized with FITC (green). The yellow color in overlay images indicates the presence of integrin β 3 on neovasculature.

Article Snippet: Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β 3 antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope.

Techniques: Single Photon Emission Computed Tomography, Immunohistochemical staining, Staining, Biomarker Assay

Journal: Theranostics

Article Title: Integrin α v β 3 -Targeted Radiotracer 99m Tc-3P-RGD 2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α v β 3 RGD 2 Therapy

doi: 10.7150/thno.6989

Figure Lengend Snippet: Microscopic images of frozen tumor slices from the xenografted MDA-MB-435 tumors (0.05 g, 0.13 g, 0.28 g, 0.55 g and 0.88 g) after immunohistochemical staining for integrin β 3 and CD31. CD31 was used as a biomarker for tumor endothelial cells. Integrin β 3 was visualized with Cy3 (red), and CD31 with FITC (green). Yellow color indicates the integrin β 3 on new blood vessels.

Article Snippet: Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β 3 antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope.

Techniques: Immunohistochemical staining, Staining, Biomarker Assay

Journal: Theranostics

Article Title: Integrin α v β 3 -Targeted Radiotracer 99m Tc-3P-RGD 2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α v β 3 RGD 2 Therapy

doi: 10.7150/thno.6989

Figure Lengend Snippet: A : Comparison of tumor volumes in vehicle and linifanib-treated in the MDA-MB-435 models. Tumor volume was determined by caliper measurements. *: p < 0.05, significantly different from the vehicle-treated group. #: p < 0.01, significantly different from the vehicle-treated group from that specific date until the end of study. B : Transverse views of selected SPECT/CT images from athymic nude mice bearing MDA-MB-435 breast tumors in the vehicle (upper panel) and linifanib-treated groups (lower panel). SPECT/CT images were obtained at -1, 1, 4 and 11 days after initiation of linifanib therapy to illustrate the early tumor response in terms of tumor uptake and integrin a v β 3 expression levels. Arrows indicate the presence of tumors.

Article Snippet: Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β 3 antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope.

Techniques: Single Photon Emission Computed Tomography, Expressing

Journal: Theranostics

Article Title: Integrin α v β 3 -Targeted Radiotracer 99m Tc-3P-RGD 2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α v β 3 RGD 2 Therapy

doi: 10.7150/thno.6989

Figure Lengend Snippet: Comparison of changes in %ID/cm 3 tumor uptake ( A ) and T/M ratios ( B ) of 99m Tc-3P-RGD 2 after linifanib treatment in three different tumor-bearing animal models (U87MG: high integrin α v β 3 expression on tumor cells and neovasculature; MDA-MB-435: moderate integrin α v β 3 expression on tumor cells and neovasculature; PC-3: low integrin α v β 3 expression on tumor cells and neovasculature). Tumor uptake values were calculated from quantification of SPECT/CT images. The %ID/cm 3 tumor uptake and T/M ratios of 99m Tc-3P-RGD 2 in the U87MG and PC-3 models were obtained from our previous report . The changes in %ID/cm 3 tumor uptake and T/M ratios were calculated by subtracting the value on a specific date (+1, +4 and +11) from the value on day -1. *: p < 0.05, significantly different from those in the U87MG and PC-3 models. # : p < 0.01, significantly different from those in the U87MG and MDA-MB-435 models.

Article Snippet: Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β 3 antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope.

Techniques: Expressing, Single Photon Emission Computed Tomography

Journal: Theranostics

Article Title: Integrin α v β 3 -Targeted Radiotracer 99m Tc-3P-RGD 2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α v β 3 RGD 2 Therapy

doi: 10.7150/thno.6989

Figure Lengend Snippet: A : Overlay images of MDA-MB-435 tumor tisues after immunohistochemical staining for integrin β 3 and CD31 in vehicle (upper panel) and linifanib-treated groups (lower panel) to illustrate the changes in integrin β 3 expression level and blood vessel density during linifanib therapy. Tumor tissues were harvested at -1, 1, 4 and 11 days after initiation of linifanib therapy. CD31 was the marker for tumor blood vessels (both mature and newly formed), which was visualized with FITC (green color). Integrin β 3 was visualized with Cy3 (red color). The yellow color in overlay images indicates the presence of integrin β 3 on new blood vessels. B : Images of selected histological slice (H&E stained) of tumor tissues from animals in the vehicle (upper panels) and linifanib-treated (lower panels) groups to illustrate vascular density changes after only one day of linifanib therapy. Red color indicates the presence of blood vessels.

Article Snippet: Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β 3 antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope.

Techniques: Immunohistochemical staining, Staining, Expressing, Marker

Journal: Theranostics

Article Title: Integrin α v β 3 -Targeted Radiotracer 99m Tc-3P-RGD 2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α v β 3 RGD 2 Therapy

doi: 10.7150/thno.6989

Figure Lengend Snippet: Linear relationship between the %ID/cm 3 tumor uptake change at days 1 (top), 4 (middle) and 11 (bottom) after linifanib therapy and the expression levels of integrin α v β 3 ( left ) and CD31 ( right ) in three animal models. The %ID/cm 3 tumor uptake values of 99m Tc-3P-RGD 2 were calculated from SPECT/CT quantification, and reported as the mean plus/minus standard error of the mean based on results from five animals (n = 5). The %ID/cm 3 tumor uptake values and integrin α v β 3 /CD31 expression levels in the U87MG and PC-3 tumors were obtained from our previous report . The %ID/cm 3 tumor uptake change was calculated by deducting the %ID/cm 3 tumor uptake of 99m Tc-3P-RGD 2 on days 1, 4 and 11 from its original value on -1 day (before linifanib therapy) in the same animal. The average %ID/cm 3 tumor uptake change is used as an indicator of tumor response to linifanib antiangiogenesis treatment.

Article Snippet: Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β 3 antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope.

Techniques: Expressing, Single Photon Emission Computed Tomography

Journal: Journal of biomedical materials research. Part A

Article Title: Platelet and endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification

doi: 10.1002/jbm.a.31888

Figure Lengend Snippet: IC 50 values for peptide interactions Concentration of free peptide corresponding to 50% inhibition (IC 50 ) of fibrinogen binding to surface immobilized α IIb β 3 integrin (left column) and to 50% inhibition of platelet aggregation (right column).

Article Snippet: Briefly, 5 μg/ml purified α IIb β 3 integrin (Enzyme Research Laboratories) was adsorbed overnight onto 96 well plates (Fisher).

Techniques: Concentration Assay, Inhibition, Binding Assay

Journal: Journal of biomedical materials research. Part A

Article Title: Platelet and endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification

doi: 10.1002/jbm.a.31888

Figure Lengend Snippet: IC 50 values for inhibition of integrin binding Concentration of free peptide corresponding to 50% inhibition of soluble ligand binding to surface immobilized integrin receptors (IC 50 , vitronectin-α V β 3 , fibronectin-α 5 β 1 , fibrinogen-α IIb β 3 ).

Article Snippet: Briefly, 5 μg/ml purified α IIb β 3 integrin (Enzyme Research Laboratories) was adsorbed overnight onto 96 well plates (Fisher).

Techniques: Inhibition, Binding Assay, Concentration Assay, Ligand Binding Assay

Journal:

Article Title: ? v ? 3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

doi:

Figure Lengend Snippet: Effect of anti-αvβ3 antibodies on endothelial cell adhesion to FGF-2-coated plastic. (A) EAhy 926 cells were incubated for 30 min at 37°C with neutralizing antisera directed to human α5β1 integrin (FNR) or αvβ3 integrin (VNR) at 1:400 or 1:200 dilution (vol/vol), respectively. Then, cells were seeded onto non-tissue culture plates coated with 20 μg/ml of FN (white bar), VN (shaded bars), or FGF-2 (black bars). The number of adherent cells was evaluated after 2 h of incubation at 37°C, subtracted from the nonspecific cell adhesion, measured onto BSA-coated wells, and expressed as percentage of cells adherent to the different substrata in the presence of nonimmune serum. Each point is the mean ± SEM of four determinations in duplicate. (B) GM 7373 cells were incubated for 30 min at 37°C with the indicated concentrations of irrelevant IgG (O) or of monoclonal anti-αvβ3 antibody (•). Then, cells were seeded onto non-tissue culture plates coated with 20 μg/ml of FGF-2. The number of adherent cells was evaluated after 2 h of incubation at 37°C, corrected for the nonspecific cell adhesion measured onto BSA-coated wells and expressed as percentage of cells adherent to the different substrata in the absence of any addition. Each point is the mean of three determinations in duplicate. SEM does not exceed 13% of the values.

Article Snippet: Anti-α v β 3 integrin antiserum was from Telios (San Diego, CA).

Techniques: Incubation

Journal:

Article Title: ? v ? 3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

doi:

Figure Lengend Snippet: Purification of αvβ3 integrin from human placenta and its interaction with immobilized FGF-2. Human term placenta extract was applied onto a wheat germ lectin-Sepharose column that was eluted with N-acetyl-d-glucosamine. Eluted proteins (a) were loaded onto a GRGDSPK-Sepharose column that was then eluted with EDTA (b). Proteins (15 and 0.5 μg/sample for a and b, respectively) were analyzed by SDS-PAGE on 8% polyacrylamide gel under reducing conditions and visualized by silver staining (A). In panel B, aliquots (0.1 μg) of purified αvβ3 integrin (corresponding to the material visualized in panel A, lane b) were analyzed by Western blotting using the indicated polyclonal antibodies. (C) Aliquots (6 μg) of purified human αvβ3 were incubated onto plastic dishes coated with FN, BSA, or FGF-2 in the absence or in the presence of soluble FN or VN (both at 75 μg/ml), or in the presence of 20 mM EDTA. At the end of incubation proteins bound to plastic were extracted and analyzed by Western blotting with anti-β3 and anti-αv antibodies. Molecular weights are in thousands.

Article Snippet: Anti-α v β 3 integrin antiserum was from Telios (San Diego, CA).

Techniques: Purification, SDS Page, Silver Staining, Western Blot, Incubation

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: TGF-β 1 mediated EMT increases cell surface α v β 3 integrin expression in NMuMG cells. NMuMG cells were incubated in the absence or presence of TGF-β 1 (5 ng/ml) for 0–36 hours and assayed for EMT. (a) Alterations in actin cytoskeletal architecture were visualized by direct rhodamine-phalloidin immunofluorescence and α v β 3 integrin by indirect immunofluorescence using anti-α v β 3 integrin (LM609) antibodies. Shown are representative images from a single experiment that was repeated twice with identical results. (b) Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to analyze expression of the α v and β 3 integrin subunits by immunoblotting. Differences in protein loading were monitored by reprobing striped blots with anti-ERK1/2 antibodies because, unlike the increase in β-actin expression observed (data not shown) ERK1/2 expression was unaltered in NMuMG cells in response to TGF-β 1 treatment. (c) β 3 Integrin deficient NMuMG cells were generated by siRNA transfection. The effects of β 3 integrin deficiency on TGF-β mediated EMT was visualized by direct FITC-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to analyze expression of the α v , β 3 , and β 1 integrin subunits by immunoblotting. Differences in protein loading were monitored by reprobing striped blots with anti-β-actin antibodies. EMT, epithelial-mesenchymal transitions; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; TGF, transforming growth factor.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Expressing, Incubation, Immunofluorescence, Western Blot, Generated, Transfection, Small Interfering RNA

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: TGF-β 1 mediated EMT induces β 3 integrin:TβR-II complex formation on the cell surface of NMuMG cells. NMuMG cells were incubated in the absence or presence of TGF-β 1 (5 ng/ml) for 0–30 hours to induce EMT. Detergent-solubilized whole cell extracts (1 mg/tube) were immunoprecipitated with (a,b) anti-β 3 or (b) anti-β 1 integrin antibodies, and subsequently immunoblotted with anti-TβR-II antibodies. Shown are representative immunoblots from a single experiment that was repeated twice with identical results. EMT, epithelial-mesenchymal transitions; TβR; TGF receptor; TGF, transforming growth factor; WCE, whole cell extracts.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Incubation, Immunoprecipitation, Western Blot

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: β 3 Integrin overexpression in NMuMG cells. NMuMG cells were engineered by bi-cistronic retroviral transduction to stably express GFP alone (control), WT β 3 integrin (WT) and GFP, or its inactive mutant D119A and GFP. (a) Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to analyze expression of the α v and β 3 integrin subunits, and of TβR-II. Differences in protein loading were monitored by immunoblotting ERK1/2, whose expression in NMuMG cells was not altered by TGF-β treatment. (b) The percentage of NMuMG cells expressing human recombinant β 3 integrin or endogenous α v integrin was determined by FACS analysis of cells incubated with either anti-human β 3 integrin or anti-mouse α v integrin antibodies. Histograms depict β 3 and α v expression only in GFP-positive NMuMG cells and are from a representative experiment that was repeated twice with similar results. (c) Iodinated TGF-β 1 was bound and chemically cross-linked to control (GFP), WT, and D119A β 3 integrin expressing cells. Cytokine:receptor complexes were isolated by immunoprecipitation with anti-TβR-II antibodies, fractionated through 7.5% SDS-PAGE gels, and immobilized electrophoretically to nitrocellulose membranes. The upper panel shows a representative phosphor image of iodinated TGF-β 1 bound to TβR-I and TβR-II. The middle panel shows that β 3 integrin was present in isolated iodinated TGF-β 1 :receptor complexes as detected by anti-β 3 integrin Western blot analysis of anti-TβR-II immunocomplexes. Differences in protein loading were monitored by probing detergent-solubilized whole cell extracts (50 μg/lane) with anti-ERK1/2 antibodies (bottom panel). BXL, binding and cross-linking; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; WT, wild type; TβR, TGF receptor; TGF, transforming growth factor; WCE, whole cell extracts.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Over Expression, Transduction, Stable Transfection, Mutagenesis, Expressing, Western Blot, Recombinant, Incubation, Isolation, Immunoprecipitation, SDS Page, Binding Assay

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: β 3 Integrin enhances MAPK activation by TGF-β in NMuMG cells. (a) Control or β 3 integrin expressing NMuMG cells were serum starved for 4 hours prior to TGF-β 1 stimulation (5 ng/ml) for 0–120 min. Afterward, the activation status of Smad2, ERK1/2, and p38 MAPK was determined by immunoblot analysis using phospho-specific antibodies. Reprobing stripped membranes with anti-ERK1/2 antibodies monitored differences in protein loading. (b) Control or β 3 integrin-expressing NMuMG cells were serum starved for 4 hours before stimulation with TGF-β 1 (5 ng/ml), prolactin (100 ng/ml), PMA (10 ng/ml), or EGF (100 ng/ml) for 30 min. Afterward, the activation status of p38 MAPK was determined by immunoblot analysis using phospho-specific antibodies. Reprobing stripped membranes with anti-pan p38 MAPK antibodies was used to monitor differences in protein loading. Accompanying graphs depict the mean (± standard error) fold increase of p38 MAPK stimulation observed in three independent experiments (* P < 0.05). EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PMA, 4α-phorbol 12-myristate 13-acetate; TGF, transforming growth factor.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: β 3 Integrin expression blocks TGF-β stimulated growth arrest but enhances TGF-β stimulated invasion and EMT in NMuMG cells. (a) Control (GFP), WT, or D119A β 3 integrin expressing NMuMG cells were stimulated with increasing concentrations of TGF-β 1 for 48 hours. Cellular DNA was radiolabeled with [ 3 H]thymidine and quantified by scintillation counting. Data are the means (± standard error) of three independent experiments presented as the percentage of [ 3 H]thymidine incorporation normalized to untreated cells. (b) Control (GFP) and β 3 integrin expressing NMuMG cells were allowed to invade through Matrigel matrices in the absence or presence of TGF-β 1 (5 ng/ml) for 36 hours. Values are the mean (± standard error) of three independent experiments presented as the percentage invasion relative to TGF-β stimulated β 3 integrin expressing NMuMG cells. (c) The cell morphology of control (GFP), WT, and D119A β 3 integrin expressing NMuMG cells was monitored by phase contrast microscopy, and alterations in actin cytoskeletal architecture was visualized by direct rhodamine-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. (d) Control (GFP), WT, or D119A β 3 integrin expressing NMuMG cells were transiently transfected with pSBE-luciferase and pCMV-β-gal cDNAs, and subsequently were stimulated with TGF-β 1 (5 ng/ml) for 24 hours. Afterward, luciferase and β-gal activities contained in detergent-solubilized cell extracts were measured. Values are the mean (± standard error) luciferase activities observed in three independent experiments normalized to maximal reporter gene expression measured in unstimulated cells. EMT, epithelial-mesenchymal transitions; GFP, green fluorescent protein; TGF, transforming growth factor; WT, wild type; SBE, Smad binding element.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Expressing, Microscopy, Immunofluorescence, Transfection, Luciferase, Binding Assay

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: Dual receptor activation induces Src mediated TβR-II tyrosine phosphorylation. (a) GFP-expressing NMuMG cells were stimulated with TGF-β 1 (5 ng/ml) for 36 hours to induce β 3 integrin expression and EMT. Afterward, the cells were dissociated and re-plated either onto plastic or vitronectin-coated wells in the absence or presence of TGF-β 1 (5 ng/ml) for 5 min. Afterward, detergent-solubilized whole cell extracts were prepared (1 mg/tube) and subsequently immunoprecipitated with anti-phosphotyrosine or anti-β 3 integrin antibodies as indicated. The presence of TβR-II in precipitated immunocomplexes was determined by Western blotting with anti-TβR-II antibodies. Differences in protein loading were monitored by immunoblotting whole cell extracts (50 mg/lane) for β 3 integrin and TβR-II. Shown are representative immunoblots from a single experiment that was repeated twice with similar results. (b) Resting WT β 3 integrin-expressing NMuMG cells were dissociated and re-plated as above. Afterward, TβR-II tyrosine phosphorylation and complex formation with β 3 integrin was determined by immunoblotting as above. Shown are representative immunoblots from a single experiment that was repeated three times with identical results. (c) Active recombinant Src (1 unit/reaction) or FAK (0.2 μg/reaction) kinases were added to protein kinase reaction mixtures containing 1 μg/tube of either kinase active (WT) or inactive (K277R) GST-TβR-II. Phosphorylation reactions were stopped after 30 min and the phosphorylation status of TβR-II was determined by immunoprecipitation of diluted protein kinase reaction mixtures with anti-TβR-II antibodies. Afterward, immobilized immunocomplexes were probed sequentially with anti-phosphotyrosine antibodies, followed by anti-TβR-II antibodies as indicated (*autophosphorylated recombinant Src; **autophosphorylated recombinant FAK). Data are from a representative experiment that was repeated three times with similar results. (d) Recombinant Src and FAK were incubated in protein kinase assay buffer for 30 min at 30°C. The data show the autophosphorylation on tyrosine residues of recombinant Src (*) and FAK (**) as determined by immunoblotting with anti-phosphotyrosine antibodies. Data are from a representative experiment that was repeated at least three times with similar results. EMT, epithelial-mesenchymal transitions; FAK, focal adhesion kinase; GST, glutathione S -transferase; TβR, TGF-β receptor; TGF, transforming growth factor; WCE, whole cell extracts; WT, wild type.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Activation Assay, Expressing, Immunoprecipitation, Western Blot, Recombinant, Incubation, Protein Kinase Assay

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: Src inhibition blocks TβR-II tyrosine phosphorylation in NMuMG cells. (a) NMuMG cells were pretreated for 60 min with either PP2 (10 μmol/l) or SU6656 (10 μmol/l). Afterward, the cells were stimulated with TGF-β 1 (5 ng/ml) for 20 min, and subsequently were lysed and immunoprecipitated (1 mg/tube) with anti-TβR-II antibodies. The resulting immunocomplexes were used to phosphorylate recombinant GST-Smad3, which was visualized by immunoblotting with anti-phospho-Smad2 antibodies. Data are from a representative experiment that was repeated three times with similar results. (b) Control or β 3 integrin expressing NMuMG cells were stimulated with TGF-β 1 (5 ng/ml) in the absence or presence the Src inhibitor PP2 (10 μmol/l), as indicated. Afterward, detergent-solubilized whole cell extracts were prepared, immunoprecipitated, and subjected to Western blot analysis either with anti-phosphotyrosine (2 mg WCE/tube) or anti-TβR-II (1 mg WCE/tube) antibodies as indicated. Differences in protein loading were monitored by probing detergent-solubilized whole cell extracts (50 μg/lane) with anti-ERK1/2 antibodies (bottom panel). (c) WT β 3 integrin expressing NMuMG cells were transiently transfected with cDNAs encoding either constitutively active (CA) or dominant negative (DN) Src. Afterward, detergent solubilized whole cell extracts were prepared, immunoprecipitated with anti-phosphotyrosine (1 mg WCE/tube), and subjected to WB analysis with anti-TβR-II antibodies. Total Src expression was monitored by probing detergent solubilized whole cell extracts (50 μg/lane) with anti-Src antibodies, whereas exogenous Src expression was confirmed by reprobing stripped membranes with anti-Myc antibodies. Differences in protein loading were monitored by probing stripped membranes with anti-TβR-II antibodies. ERK, extracellular signal-regulated kinase; GST, glutathione S -transferase; IP, immunoprecipitated; PY, phosphotyrosine; TβR, TGF-β receptor; TGF, transforming growth factor; WB, Western blot; WCE, whole cell extracts; WT, wild type.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Inhibition, Immunoprecipitation, Recombinant, Western Blot, Expressing, Transfection, Dominant Negative Mutation

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: Src inhibition blocks β 3 integrin and TGF-β mediated MAPK activation, EMT, and invasion in NMuMG cells. (a) Control or β 3 integrin expressing NMuMG cells were serum starved for 4 hours before TGF-β 1 (5 ng/ml) stimulation in the absence or presence of either PP2 (10 μmol/l) or its inactive counterpart PP3 (10 μmol/l). Afterward, the activation status of ERK1/2 and p38 MAPK was determined by immunoblot analysis using phospho-specific antibodies. Re-probing stripped membranes with anti-ERK1/2 antibodies monitored differences in protein loading. Data are from a representative experiment that was repeated twice with similar results. (b) β 3 Integrin expressing NMuMG cells were stimulated with TGF-β 1 (5 ng/ml) for 36 hours in the absence or presence of PP2 (10 μmol/l). Afterward, alterations in actin cytoskeletal architecture were visualized by direct rhodamine-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. (c) Control (GFP) or DN Src-expressing NMuMG cells were stimulated with TGF-β 1 (5 ng/ml) for 24 hours. Afterward, alterations in actin cytoskeletal architecture were visualized by direct rhodamine-phalloidin immunofluorescence. Total Src expression was monitored by probing detergent-solubilized whole cell extracts (50 μg/lane) with anti-Src antibodies, whereas exogenous Src expression was visualized using anti-Myc antibodies. Immunoblotting stripped membranes with anti-β-actin antibodies served to monitor differences in protein loading. Shown are representative images from a single experiment that was repeated twice with identical results. (d) Control (GFP), DN Src, CA Src, β 3 integrin, and β 3 integrin/DN Src expressing NMuMG cells were allowed to invade through Matrigel matrices in the absence or presence of TGF-β 1 (5 ng/ml) for 36 hours. Values are the mean (± standard error) of three independent experiments presented as the number of cells invaded per well. (e) Src-deficient NMuMG cells were generated by siRNA transfection. The effects of Src deficiency on TGF-β mediated EMT were visualized by direct FITC-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to monitor Src expression by immunoblotting with anti-Src antibodies. Differences in protein loading were monitored by re-probing striped blots with anti-β-actin antibodies. CA, constitutively active; DN, dominant negative; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; TGF, transforming growth factor; WB, Western blot; WT, wild type.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Immunofluorescence, Generated, Transfection, Dominant Negative Mutation

Journal: Breast Cancer Research

Article Title: β 3 Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

doi: 10.1186/bcr1524

Figure Lengend Snippet: β 3 Integrin expression increases in malignant human MECs and regulates invasion stimulated by TGF-β. (a) Detergent-solubilized whole cell extracts (100 μg/lane) of MCF10A and MCF10CA1a cells were prepared to analyze expression of the β 3 integrin subunit and FAK by immunoblotting. Differences in protein loading were monitored by probing striped blots with anti-TβR-II antibodies. Control (GFP) and β 3 integrin expressing (b) MCF10A cells or (c) MCF10CA1a cells were allowed to invade through Matrigel matrices in the absence or presence of TGF-β 1 (5 ng/ml) for 36 hours. Values are the mean (± standard error) of three independent experiments presented as the percentage invasion relative to TGF-β stimulated WT β 3 integrin expressing cells. FAK, focal adhesion kinase; MEC, mammary epithelial cell; TβR, TGF-β receptor; TGF, transforming growth factor; WT, wild type.

Article Snippet: Afterward, 1 × 10 6 cells were trypsinized, washed, and incubated in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumen in phosphate-buffered saline [PBS]) supplemented with a 1:20 dilution of either PE-conjugated anti-mouse α v integrin (BD Pharmingen, San Diego, CA, USA) or PE-conjugated anti-human β 3 integrin antibodies (BD Pharmingen).

Techniques: Expressing, Western Blot