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R&D Systems mouse tgf β2 neutralizing antibody

Cytokine level is affected in muscular laminopathies. (A) Levels of <t>TGF</t> <t>β2</t> in sera of healthy donors (control), patients affected by muscular laminopathies ( LMNA) and patients affected by other neuromuscular diseases, including Duchenne Muscular dystrophy, Becker muscular dystrophy and Myotonic Dystrophy (others). (B) Levels of TGF β2, IL6, IL17 and FGF-b in sera of controls and LMNA symptomatic patients. Based on cytokine expression pattern, sera from LMNA symptomatic patients are divided in three subgroups (LMNA1, LMNA2, LMNA3); the percentage of patients in each subgroup out of all examined LMNA patients is reported. (C) Levels of TGF β1, TGF β2, TGF β3 in sera of controls and LMNA patients. (D) Representative image of immunofluorescence detection of TGF β2 (green) in control and LMNA muscle tissue (in this case R190Q/R249Q heterozygous compound mutation in LMNA was determined in the EDMD2 patient). Nuclei are stained with DAPI. Bar: 10µm. (E) Scheme of LMNA mutations detected in laminopathic patients examined in this study. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p<0.01) or triple asterisk (p< 0.001).

Mouse Tgf β2 Neutralizing Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes"

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

Journal: Nucleus

doi: 10.1080/19491034.2018.1467722

Cytokine level is affected in muscular laminopathies. (A) Levels of TGF β2 in sera of healthy donors (control), patients affected by muscular laminopathies ( LMNA) and patients affected by other neuromuscular diseases, including Duchenne Muscular dystrophy, Becker muscular dystrophy and Myotonic Dystrophy (others). (B) Levels of TGF β2, IL6, IL17 and FGF-b in sera of controls and LMNA symptomatic patients. Based on cytokine expression pattern, sera from LMNA symptomatic patients are divided in three subgroups (LMNA1, LMNA2, LMNA3); the percentage of patients in each subgroup out of all examined LMNA patients is reported. (C) Levels of TGF β1, TGF β2, TGF β3 in sera of controls and LMNA patients. (D) Representative image of immunofluorescence detection of TGF β2 (green) in control and LMNA muscle tissue (in this case R190Q/R249Q heterozygous compound mutation in LMNA was determined in the EDMD2 patient). Nuclei are stained with DAPI. Bar: 10µm. (E) Scheme of LMNA mutations detected in laminopathic patients examined in this study. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p<0.01) or triple asterisk (p< 0.001).

Figure Legend Snippet: Cytokine level is affected in muscular laminopathies. (A) Levels of TGF β2 in sera of healthy donors (control), patients affected by muscular laminopathies ( LMNA) and patients affected by other neuromuscular diseases, including Duchenne Muscular dystrophy, Becker muscular dystrophy and Myotonic Dystrophy (others). (B) Levels of TGF β2, IL6, IL17 and FGF-b in sera of controls and LMNA symptomatic patients. Based on cytokine expression pattern, sera from LMNA symptomatic patients are divided in three subgroups (LMNA1, LMNA2, LMNA3); the percentage of patients in each subgroup out of all examined LMNA patients is reported. (C) Levels of TGF β1, TGF β2, TGF β3 in sera of controls and LMNA patients. (D) Representative image of immunofluorescence detection of TGF β2 (green) in control and LMNA muscle tissue (in this case R190Q/R249Q heterozygous compound mutation in LMNA was determined in the EDMD2 patient). Nuclei are stained with DAPI. Bar: 10µm. (E) Scheme of LMNA mutations detected in laminopathic patients examined in this study. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p<0.01) or triple asterisk (p< 0.001).

Techniques Used: Control, Expressing, Immunofluorescence, Mutagenesis, Staining, Standard Deviation

TGF β2 level is increased in laminopathic mice. (a) Levels of TGF β2 in sera of WT mice (n = 6) and Lmna H222P/H222P mice ( Lmna H222P ) (n = 5). (b) Levels of TGF β2 secreted by fibroblasts isolated from WT mice ( Lmna +/+ ), mice bearing heterozigous ( Lmna +/− ) or homozygous ( Lmna −/− ) null mutation in Lmna gene and maintained in culture for 5 days. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05) or double asterisk (p< 0.01).

Figure Legend Snippet: TGF β2 level is increased in laminopathic mice. (a) Levels of TGF β2 in sera of WT mice (n = 6) and Lmna H222P/H222P mice ( Lmna H222P ) (n = 5). (b) Levels of TGF β2 secreted by fibroblasts isolated from WT mice ( Lmna +/+ ), mice bearing heterozigous ( Lmna +/− ) or homozygous ( Lmna −/− ) null mutation in Lmna gene and maintained in culture for 5 days. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05) or double asterisk (p< 0.01).

Techniques Used: Isolation, Mutagenesis, Standard Deviation

EDMD2 fibroblast medium induces TGF β2-dependent fibrogenic conversion in NHM cultures. (A) TGF β2 secretion in control and EDMD2 fibroblast culture medium. (B) Quantification of myofibroblasts in cultures of control human myoblasts maintained in medium conditioned by control (control) or EDMD2 (EDMD2) fibroblasts or treated with TGF β2 (control + TGF β2). The number of myofibroblasts was determined by counting alpha-SMA positive mononucleated cells (200 cells/sample were counted in three independent experiments). (C) Immunofluorescence staining of collagen I (green) and desmin (red) in cycling and differentiated NHM cultured in presence of medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGFβ2) or not (NT) with anti-TGF β2 neutralizing antibodies. Nuclei were stained with DAPI. Bar: 10µm. (D) Immunofluorescence staining of ED-fibronectin (red) in cryosections of muscle tissue isolated from healthy donors (control), EDMD2 patients (EDMD2) or Becker muscular dystrophy patients (BMD). Nuclei were stained with DAPI. BMD tissue was used as positive control. In control muscle, ED-fibronectin is restricted to the area surrounding blood vessels. Bar: 10µm. (E) Quantitative analysis of proliferating cells in NHM cultures maintained in medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGF β2) or not (NT) with anti-TGF β2 neutralizing antibodies. The number of proliferating cells was determined by flow cytometry. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by double asterisk (p<0.01) or triple asterisk (p< 0.001).

Figure Legend Snippet: EDMD2 fibroblast medium induces TGF β2-dependent fibrogenic conversion in NHM cultures. (A) TGF β2 secretion in control and EDMD2 fibroblast culture medium. (B) Quantification of myofibroblasts in cultures of control human myoblasts maintained in medium conditioned by control (control) or EDMD2 (EDMD2) fibroblasts or treated with TGF β2 (control + TGF β2). The number of myofibroblasts was determined by counting alpha-SMA positive mononucleated cells (200 cells/sample were counted in three independent experiments). (C) Immunofluorescence staining of collagen I (green) and desmin (red) in cycling and differentiated NHM cultured in presence of medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGFβ2) or not (NT) with anti-TGF β2 neutralizing antibodies. Nuclei were stained with DAPI. Bar: 10µm. (D) Immunofluorescence staining of ED-fibronectin (red) in cryosections of muscle tissue isolated from healthy donors (control), EDMD2 patients (EDMD2) or Becker muscular dystrophy patients (BMD). Nuclei were stained with DAPI. BMD tissue was used as positive control. In control muscle, ED-fibronectin is restricted to the area surrounding blood vessels. Bar: 10µm. (E) Quantitative analysis of proliferating cells in NHM cultures maintained in medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGF β2) or not (NT) with anti-TGF β2 neutralizing antibodies. The number of proliferating cells was determined by flow cytometry. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by double asterisk (p<0.01) or triple asterisk (p< 0.001).

Techniques Used: Control, Immunofluorescence, Staining, Cell Culture, Isolation, Positive Control, Flow Cytometry, Standard Deviation

EDMD2 fibroblast medium inhibits differentiation of NHM through TGF β2. (A) Immunofluorescence staining of myogenin (red) and caveolin 3 (green) in C2C12 myoblasts cultured in presence of control or EDMD2 fibroblast medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. Nuclei are counterstained with DAPI. Bar: 10µm. (B) Percentage of differentiated cells in C2C12 myoblast cultures conditioned by control or EDMD2 medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. (C) Percentage of differentiated NHM conditioned by EDMD2 medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05) or double asterisk (p< 0.01).

Figure Legend Snippet: EDMD2 fibroblast medium inhibits differentiation of NHM through TGF β2. (A) Immunofluorescence staining of myogenin (red) and caveolin 3 (green) in C2C12 myoblasts cultured in presence of control or EDMD2 fibroblast medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. Nuclei are counterstained with DAPI. Bar: 10µm. (B) Percentage of differentiated cells in C2C12 myoblast cultures conditioned by control or EDMD2 medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. (C) Percentage of differentiated NHM conditioned by EDMD2 medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05) or double asterisk (p< 0.01).

Techniques Used: Immunofluorescence, Staining, Cell Culture, Control, Standard Deviation

TGF β2 from EDMD2 serum induces fibrosis markers in normal human tenocytes. (A) Immunofluorescence staining of alpha-SMA in normal human tenocytes cultured in the presence of control serum, control serum + TGF β2, EDMD2 serum or EDMD2 serum + anti-TGF β2. Nuclei were counterstained with DAPI. Bar, 20 μm. Quantitative analysis of mean fluorescence intensity of alpha-SMA is reported in the graph. (B) Western blot analysis of ED-fibronectin, tenomodulin and alpha-SMA in control tenocytes exposed to control serum, control serum + TGF β2 or EDMD2 serum. Densitometric analysis of immunoblotted bands is reported in the graphs. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p< 0.01) or triple asterisk (p< 0.001).

Figure Legend Snippet: TGF β2 from EDMD2 serum induces fibrosis markers in normal human tenocytes. (A) Immunofluorescence staining of alpha-SMA in normal human tenocytes cultured in the presence of control serum, control serum + TGF β2, EDMD2 serum or EDMD2 serum + anti-TGF β2. Nuclei were counterstained with DAPI. Bar, 20 μm. Quantitative analysis of mean fluorescence intensity of alpha-SMA is reported in the graph. (B) Western blot analysis of ED-fibronectin, tenomodulin and alpha-SMA in control tenocytes exposed to control serum, control serum + TGF β2 or EDMD2 serum. Densitometric analysis of immunoblotted bands is reported in the graphs. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p< 0.01) or triple asterisk (p< 0.001).

Techniques Used: Immunofluorescence, Staining, Cell Culture, Control, Fluorescence, Western Blot, Standard Deviation

TGF β2 affects the AKT/mTOR pathway in EDMD2. Western blot analysis of p-mTOR, mTOR, Thr308 Akt, Ser473 Akt, Akt and actin performed in (A) control myoblasts (control), EDMD2 myoblasts (EDMD2) and EDMD2 myoblasts treated with anti-TGF β2 neutralizing antibody (EDMD2 + anti-TGF β2) or (B) control fibroblasts (control), EDMD2 fibroblasts (EDMD2) and EDMD2 fibroblasts treated with anti-TGF β2 neutralizing antibody (EDMD2 + anti-TGF β2). Actin bands are shown as protein loading control. Molecular weight markers are reported in kDa. Densitometric analysis of immunoblotted bands normalized on actin is reported in the graphs. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p< 0.01) or triple asterisk (p< 0.001).

Figure Legend Snippet: TGF β2 affects the AKT/mTOR pathway in EDMD2. Western blot analysis of p-mTOR, mTOR, Thr308 Akt, Ser473 Akt, Akt and actin performed in (A) control myoblasts (control), EDMD2 myoblasts (EDMD2) and EDMD2 myoblasts treated with anti-TGF β2 neutralizing antibody (EDMD2 + anti-TGF β2) or (B) control fibroblasts (control), EDMD2 fibroblasts (EDMD2) and EDMD2 fibroblasts treated with anti-TGF β2 neutralizing antibody (EDMD2 + anti-TGF β2). Actin bands are shown as protein loading control. Molecular weight markers are reported in kDa. Densitometric analysis of immunoblotted bands normalized on actin is reported in the graphs. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p< 0.01) or triple asterisk (p< 0.001).

Techniques Used: Western Blot, Control, Molecular Weight, Standard Deviation

Image Search Results

Journal: Nucleus

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

doi: 10.1080/19491034.2018.1467722

Figure Lengend Snippet: Cytokine level is affected in muscular laminopathies. (A) Levels of TGF β2 in sera of healthy donors (control), patients affected by muscular laminopathies ( LMNA) and patients affected by other neuromuscular diseases, including Duchenne Muscular dystrophy, Becker muscular dystrophy and Myotonic Dystrophy (others). (B) Levels of TGF β2, IL6, IL17 and FGF-b in sera of controls and LMNA symptomatic patients. Based on cytokine expression pattern, sera from LMNA symptomatic patients are divided in three subgroups (LMNA1, LMNA2, LMNA3); the percentage of patients in each subgroup out of all examined LMNA patients is reported. (C) Levels of TGF β1, TGF β2, TGF β3 in sera of controls and LMNA patients. (D) Representative image of immunofluorescence detection of TGF β2 (green) in control and LMNA muscle tissue (in this case R190Q/R249Q heterozygous compound mutation in LMNA was determined in the EDMD2 patient). Nuclei are stained with DAPI. Bar: 10µm. (E) Scheme of LMNA mutations detected in laminopathic patients examined in this study. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p<0.01) or triple asterisk (p< 0.001).

Article Snippet: In order to neutralize TGF β2, human myoblast and C2C12 cultures were treated for 72 hours with human TGF β2 neutralizing antibody (ab66045, Abcam, Cambridge, UK) at the concentration of 0,03 μg/ml or mouse TGF β2 neutralizing antibody at the concentration of 0,01 μg/ml (MAB7346, R&D System, Abington, UK), respectively.

Techniques: Control, Expressing, Immunofluorescence, Mutagenesis, Staining, Standard Deviation

Journal: Nucleus

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

doi: 10.1080/19491034.2018.1467722

Figure Lengend Snippet: TGF β2 level is increased in laminopathic mice. (a) Levels of TGF β2 in sera of WT mice (n = 6) and Lmna H222P/H222P mice ( Lmna H222P ) (n = 5). (b) Levels of TGF β2 secreted by fibroblasts isolated from WT mice ( Lmna +/+ ), mice bearing heterozigous ( Lmna +/− ) or homozygous ( Lmna −/− ) null mutation in Lmna gene and maintained in culture for 5 days. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05) or double asterisk (p< 0.01).

Techniques: Isolation, Mutagenesis, Standard Deviation

Journal: Nucleus

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

doi: 10.1080/19491034.2018.1467722

Figure Lengend Snippet: EDMD2 fibroblast medium induces TGF β2-dependent fibrogenic conversion in NHM cultures. (A) TGF β2 secretion in control and EDMD2 fibroblast culture medium. (B) Quantification of myofibroblasts in cultures of control human myoblasts maintained in medium conditioned by control (control) or EDMD2 (EDMD2) fibroblasts or treated with TGF β2 (control + TGF β2). The number of myofibroblasts was determined by counting alpha-SMA positive mononucleated cells (200 cells/sample were counted in three independent experiments). (C) Immunofluorescence staining of collagen I (green) and desmin (red) in cycling and differentiated NHM cultured in presence of medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGFβ2) or not (NT) with anti-TGF β2 neutralizing antibodies. Nuclei were stained with DAPI. Bar: 10µm. (D) Immunofluorescence staining of ED-fibronectin (red) in cryosections of muscle tissue isolated from healthy donors (control), EDMD2 patients (EDMD2) or Becker muscular dystrophy patients (BMD). Nuclei were stained with DAPI. BMD tissue was used as positive control. In control muscle, ED-fibronectin is restricted to the area surrounding blood vessels. Bar: 10µm. (E) Quantitative analysis of proliferating cells in NHM cultures maintained in medium conditioned by control or EDMD2 fibroblasts, treated (anti-TGF β2) or not (NT) with anti-TGF β2 neutralizing antibodies. The number of proliferating cells was determined by flow cytometry. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by double asterisk (p<0.01) or triple asterisk (p< 0.001).

Techniques: Control, Immunofluorescence, Staining, Cell Culture, Isolation, Positive Control, Flow Cytometry, Standard Deviation

Journal: Nucleus

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

doi: 10.1080/19491034.2018.1467722

Figure Lengend Snippet: EDMD2 fibroblast medium inhibits differentiation of NHM through TGF β2. (A) Immunofluorescence staining of myogenin (red) and caveolin 3 (green) in C2C12 myoblasts cultured in presence of control or EDMD2 fibroblast medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. Nuclei are counterstained with DAPI. Bar: 10µm. (B) Percentage of differentiated cells in C2C12 myoblast cultures conditioned by control or EDMD2 medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. (C) Percentage of differentiated NHM conditioned by EDMD2 medium. Data from samples left untreated (NT) or treated with anti-TGF β2 antibody (anti-TGF β2) are reported. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05) or double asterisk (p< 0.01).

Techniques: Immunofluorescence, Staining, Cell Culture, Control, Standard Deviation

Journal: Nucleus

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

doi: 10.1080/19491034.2018.1467722

Figure Lengend Snippet: TGF β2 from EDMD2 serum induces fibrosis markers in normal human tenocytes. (A) Immunofluorescence staining of alpha-SMA in normal human tenocytes cultured in the presence of control serum, control serum + TGF β2, EDMD2 serum or EDMD2 serum + anti-TGF β2. Nuclei were counterstained with DAPI. Bar, 20 μm. Quantitative analysis of mean fluorescence intensity of alpha-SMA is reported in the graph. (B) Western blot analysis of ED-fibronectin, tenomodulin and alpha-SMA in control tenocytes exposed to control serum, control serum + TGF β2 or EDMD2 serum. Densitometric analysis of immunoblotted bands is reported in the graphs. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p< 0.01) or triple asterisk (p< 0.001).

Techniques: Immunofluorescence, Staining, Cell Culture, Control, Fluorescence, Western Blot, Standard Deviation

Journal: Nucleus

Article Title: Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

doi: 10.1080/19491034.2018.1467722

Figure Lengend Snippet: TGF β2 affects the AKT/mTOR pathway in EDMD2. Western blot analysis of p-mTOR, mTOR, Thr308 Akt, Ser473 Akt, Akt and actin performed in (A) control myoblasts (control), EDMD2 myoblasts (EDMD2) and EDMD2 myoblasts treated with anti-TGF β2 neutralizing antibody (EDMD2 + anti-TGF β2) or (B) control fibroblasts (control), EDMD2 fibroblasts (EDMD2) and EDMD2 fibroblasts treated with anti-TGF β2 neutralizing antibody (EDMD2 + anti-TGF β2). Actin bands are shown as protein loading control. Molecular weight markers are reported in kDa. Densitometric analysis of immunoblotted bands normalized on actin is reported in the graphs. Means ± standard deviation are shown in graphs. Statistically significant differences are indicated by an asterisk (p<0.05), double asterisk (p< 0.01) or triple asterisk (p< 0.001).

Techniques: Western Blot, Control, Molecular Weight, Standard Deviation

The CXCL12-CXCR4/CXCR7 axis in the inducible-invasive phenotype. (A) Levels of CXCL12 expressed in pgs/ml in the CMs of the BrC cell lines. (B) MCF-7 and T47D cells were cultured with CM from HA-BrC cell lines and controls, and expression of chemokine receptors was analyzed by flow cytometry. (C) CXCL12 or (D) CMs as chemoattractants. (E) CMs from the HA BrC cells were used as chemoattractants and 0.5 µ g/ml of an anti-CXCL12 neutralizing antibody was added to the CMs. Invasive cells were quantified after 24 h (magnification of ×100). The integrated optical density (IOD) values of invading cells were plotted. Data represent the mean ± SEM from 3 independent experiments.; * P<0.05, ** P<0.01 and *** P<0.001. Scale bars indicate 100 µ m.

Journal: International Journal of Oncology

Article Title: Evidence of lateral transmission of aggressive features between different types of breast cancer cells

doi: 10.3892/ijo.2017.4128

Figure Lengend Snippet: The CXCL12-CXCR4/CXCR7 axis in the inducible-invasive phenotype. (A) Levels of CXCL12 expressed in pgs/ml in the CMs of the BrC cell lines. (B) MCF-7 and T47D cells were cultured with CM from HA-BrC cell lines and controls, and expression of chemokine receptors was analyzed by flow cytometry. (C) CXCL12 or (D) CMs as chemoattractants. (E) CMs from the HA BrC cells were used as chemoattractants and 0.5 µ g/ml of an anti-CXCL12 neutralizing antibody was added to the CMs. Invasive cells were quantified after 24 h (magnification of ×100). The integrated optical density (IOD) values of invading cells were plotted. Data represent the mean ± SEM from 3 independent experiments.; * P<0.05, ** P<0.01 and *** P<0.001. Scale bars indicate 100 µ m.

Article Snippet: To neutralize the biological activity of TGF-β of the HA-CMs, 2 µ g/ml of the rabbit anti-human TGF-β1, anti-β2, and anti-β3 neutralizing antibody (anti-TGF-β, R&D Systems, Inc., ref. MAB1835) were added according to the guideline provided by the manufacturer.

Techniques: Cell Culture, Expressing, Flow Cytometry

The inducible-invasive phenotype is TGF-β independent. (A) The concentration expressed in pgs/ml of TGF-β was measured in the CM from all BrC cell lines and data were plotted. The inducible-invasive phenotype of MCF-7 and T47D cells was activated with HA-BrC CMs in the presence of 2 µ g/ml of neutralizing anti-TGF-β. After 72 h of culture, (B) EMT markers were analyzed by IF and IODs of E-cadherin expression were quantified and plotted. (C) Invasion assays were performed. Left panels show representative images of invading cells and right plots show the number of invading cells. (D) Analysis of EMT markers and invasion assays of NA-BrC cell lines treated with increasing concentrations of TGF-β. Representative images are shown. Data represents the mean ± SEM from 3 independent experiments; *** P<0.001; ns, non-significant. Scale bars indicate 100 µ m, magnification of ×400 for IF and ×100 for invasion assays.

Journal: International Journal of Oncology

Article Title: Evidence of lateral transmission of aggressive features between different types of breast cancer cells

doi: 10.3892/ijo.2017.4128

Figure Lengend Snippet: The inducible-invasive phenotype is TGF-β independent. (A) The concentration expressed in pgs/ml of TGF-β was measured in the CM from all BrC cell lines and data were plotted. The inducible-invasive phenotype of MCF-7 and T47D cells was activated with HA-BrC CMs in the presence of 2 µ g/ml of neutralizing anti-TGF-β. After 72 h of culture, (B) EMT markers were analyzed by IF and IODs of E-cadherin expression were quantified and plotted. (C) Invasion assays were performed. Left panels show representative images of invading cells and right plots show the number of invading cells. (D) Analysis of EMT markers and invasion assays of NA-BrC cell lines treated with increasing concentrations of TGF-β. Representative images are shown. Data represents the mean ± SEM from 3 independent experiments; *** P<0.001; ns, non-significant. Scale bars indicate 100 µ m, magnification of ×400 for IF and ×100 for invasion assays.

Techniques: Concentration Assay, Expressing

(A–D) The role of pro-inflammatory mediators in the inducible-invasive phenotype. (A) Milliplex assays were performed to determine the concentration of pro-inflammatory mediators and metalloproteinases (expressed in pgs/ml) in all the CMs; only analytes exhibiting significant differences between the CMs of NA- and HA-BrC cells are shown. MCF-7 and T47D cells were cultured with 100 ng/ml of any of the following: G-CSF, GM-CSF, IL-8 or MCP-1 for 72 h. (B) Analysis of the EMT marker E-cadherin by IF. (C) Plots of the analysis of CXCR4 and CXCR7 chemokine receptor expression by flow cytometry. Invasion assays using CXCL12 (D) as chemoattractant. Representative images and plots of resulting data are shown. Data represent the mean ± SEM from 3 independent experiments; * P<0.05, ** P<0.01 and *** P<0.001. In the panels of T47D cells of (D), IL-8 was significantly different than the other cytokines ( * P<0.05). Scale bars indicate 100 µ m and magnification, ×100. (E and F) The role of pro-inflammatory mediators in the inducible-invasive phenotype. Invasion assays using fetal bovine serum (FBS) (E) as chemoattractant. (F) A Milliplex assay was performed to determine the sera concentration of the pro-inflammatory mediators and metalloproteinases of interest in BrC patients and controls. Representative images and plots of resulting data are shown. Data represent the mean ± SEM from 2 independent experiments; *** P<0.001. Only two duplicates were analyzed (F). Scale bars indicate 100 µ m and magnification, ×100.

Journal: International Journal of Oncology

Article Title: Evidence of lateral transmission of aggressive features between different types of breast cancer cells

doi: 10.3892/ijo.2017.4128

Figure Lengend Snippet: (A–D) The role of pro-inflammatory mediators in the inducible-invasive phenotype. (A) Milliplex assays were performed to determine the concentration of pro-inflammatory mediators and metalloproteinases (expressed in pgs/ml) in all the CMs; only analytes exhibiting significant differences between the CMs of NA- and HA-BrC cells are shown. MCF-7 and T47D cells were cultured with 100 ng/ml of any of the following: G-CSF, GM-CSF, IL-8 or MCP-1 for 72 h. (B) Analysis of the EMT marker E-cadherin by IF. (C) Plots of the analysis of CXCR4 and CXCR7 chemokine receptor expression by flow cytometry. Invasion assays using CXCL12 (D) as chemoattractant. Representative images and plots of resulting data are shown. Data represent the mean ± SEM from 3 independent experiments; * P<0.05, ** P<0.01 and *** P<0.001. In the panels of T47D cells of (D), IL-8 was significantly different than the other cytokines ( * P<0.05). Scale bars indicate 100 µ m and magnification, ×100. (E and F) The role of pro-inflammatory mediators in the inducible-invasive phenotype. Invasion assays using fetal bovine serum (FBS) (E) as chemoattractant. (F) A Milliplex assay was performed to determine the sera concentration of the pro-inflammatory mediators and metalloproteinases of interest in BrC patients and controls. Representative images and plots of resulting data are shown. Data represent the mean ± SEM from 2 independent experiments; *** P<0.001. Only two duplicates were analyzed (F). Scale bars indicate 100 µ m and magnification, ×100.

Techniques: Concentration Assay, Cell Culture, Marker, Expressing, Flow Cytometry

(A–D) The induced-invasive phenotype correlates with acquisition of stemness markers. Analysis of the basal expression levels of stemness markers: CD44 by flow cytometry (A), and of Oct-4 and Sox-2 by immunofluorescence (IF) (B). Rrepresentative images are shown. Scale bars indicate 100 µ m (B). Magnification, ×400. Analysis of the expression levels of CD44 after induction of the invasive phenotype in MCF-7 (C) and T47D (D) cells. The upper panel shows CD44 expression and the lower panel shows plots of the frequency of CD44 + cells and the CD44 MFI. Data represent the mean ± SEM from 3 independent experiments, representative images are shown. ** P<0.01 and *** P<0.001. (E–I) The induced-invasive phenotype correlates with acquisition of stemness markers. Analysis of Oct-4 and Sox-2 IODs is shown for MCF-7 (E) and T47D (F) cells. (G) Sox-2 was also examined by FACS; the upper panels show representative images of cell density plots, while the frequency of Sox-2 positive cells and the MFI of Sox-2 expression are graphed below. (H) The intrinsic sphere forming efficiency of the BrC cell lines was analyzed in ultra-low attachment plates, and (I) after induction of the invasive phenotype in MCF-7 and T47D cells. Plots of the frequency and size of the tumorspheres are shown. Data represent the mean ± SEM from 3 independent experiments, representative images are shown. * P<0.05, ** P<0.01 and *** P<0.001. Scale bars indicate 50 µ m for MCF-7 and T47D, and 100 µ m for HS578T and MDA-MB-231 tumorspheres (H and I). Magnification, ×400.

Journal: International Journal of Oncology

Article Title: Evidence of lateral transmission of aggressive features between different types of breast cancer cells

doi: 10.3892/ijo.2017.4128

Figure Lengend Snippet: (A–D) The induced-invasive phenotype correlates with acquisition of stemness markers. Analysis of the basal expression levels of stemness markers: CD44 by flow cytometry (A), and of Oct-4 and Sox-2 by immunofluorescence (IF) (B). Rrepresentative images are shown. Scale bars indicate 100 µ m (B). Magnification, ×400. Analysis of the expression levels of CD44 after induction of the invasive phenotype in MCF-7 (C) and T47D (D) cells. The upper panel shows CD44 expression and the lower panel shows plots of the frequency of CD44 + cells and the CD44 MFI. Data represent the mean ± SEM from 3 independent experiments, representative images are shown. ** P<0.01 and *** P<0.001. (E–I) The induced-invasive phenotype correlates with acquisition of stemness markers. Analysis of Oct-4 and Sox-2 IODs is shown for MCF-7 (E) and T47D (F) cells. (G) Sox-2 was also examined by FACS; the upper panels show representative images of cell density plots, while the frequency of Sox-2 positive cells and the MFI of Sox-2 expression are graphed below. (H) The intrinsic sphere forming efficiency of the BrC cell lines was analyzed in ultra-low attachment plates, and (I) after induction of the invasive phenotype in MCF-7 and T47D cells. Plots of the frequency and size of the tumorspheres are shown. Data represent the mean ± SEM from 3 independent experiments, representative images are shown. * P<0.05, ** P<0.01 and *** P<0.001. Scale bars indicate 50 µ m for MCF-7 and T47D, and 100 µ m for HS578T and MDA-MB-231 tumorspheres (H and I). Magnification, ×400.

Techniques: Expressing, Flow Cytometry, Immunofluorescence

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