rabbit anti tgf β3 antibody  (Bioss)

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

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.

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: Concentration Assay, Expressing

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.

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

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.

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: Expressing, Flow Cytometry, Immunofluorescence