Journal: Genomics, Proteomics & Bioinformatics

Article Title: Screening Preeclamptic Cord Plasma for Proteins Associated with Decreased Breast Cancer Susceptibility

doi: 10.1016/j.gpb.2013.09.009

Figure Lengend Snippet: Validation of protein expression by Western blot analysis Western blot analysis was performed to detect the presence of alpha-fetoprotein (AFP), complement C5 (C5), pigment epithelium-derived factor (PEDF), and complement factor H (factor H) in normotensive (N1, N2 and N3) and preeclamptic (PE1, PE2 and PE3) umbilical cord blood samples. A . A Coomassie blue-stained SDS–PAGE gel showed equal loading of 10 μg protein per lane of all samples tested. B . Western blot analysis for AFP showed a significant association between the densitometric intensities of the bands with the corresponding normalized protein quantity ( R 2 = 0.91, P = 0.003 significant at ∗∗ P < 0.01, linear regression analysis) but a nonsignificant ( P = 0.6, t -test) up-regulation of AFP in PE when compared to N samples. C . Western blot analysis and quantification densitometric analysis showed that expression of PEDF was significantly up-regulated in PE when compared to N samples ( P = 0.006 significant at ∗∗ P < 0.01, t -test), whereas up-regulation for C5 expression and down-regulation for Factor H expression in PE plasma were not significant ( P = 0.3 and 0.2, respectively, t -test). Error bars represent standard error of the mean (SEM).

Article Snippet: After washing with TBS-T, the appropriate blot was incubated overnight at 4 °C with one of the following antibodies: anti-human Factor H (goat, 1:2000; AF4779, R&D Systems, Minneapolis, MN), anti-human AFP antibodies (chicken, 1:2000; AF1369, R&D Systems), mouse anti-human C5 (mouse, 1:500; sc-70476, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-human PEDF (mouse, 1:500; sc-53921, Santa Cruz Biotechnology).

Techniques: Biomarker Discovery, Expressing, Western Blot, Derivative Assay, Staining, SDS Page, Clinical Proteomics

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Immunohistochemical analysis of E-cadherin, vimentin, Snail and NF-κB in invasive ductal breast cancer (IDC) and normal breast samples (original magnification, ×400). (A) Immunostaining of pigment epithelium-derived factor (PEDF) mainly in the cytoplasm of certain epithelial cells (yellow-brown granules indicated by the red arrow). (B)Immunostaining of E-cadherin in the cell membranes of normal breast tissues (yellow-brown granules indicated by the red arrow). (C) Immunostaining of vimentin in the normal breast tissues. (D) Immunostaining of nuclear factor κβ (NF-κβ) in the normal breast tissues. (E) Immunostaining of Snail in the normal breast tissues. (F) Immunostaining of PEDF in invasive ductal breast cancer samples. (G) Immunostaining of E-cadherin in invasive ductal breast cancer samples. (H) Immunostaining of vimentin in the cytoplasm of certain epithelial cells (yellow-brown granules indicated by the red arrow). (I) Immunostaining of NF-κβ in the nuclei and cytoplasm of cancer cells in a positive specimen (yellow-brown granules indicated by a red arrow). (J) Immunostaining of Snail in the cytoplasm of IDC cells in a positive specimen (yellow-brown granules indicated by a red arrow).

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques: Immunohistochemical staining, Immunostaining, Derivative Assay

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Correlation between PEDF, E-cadherin, vimentin, Snail and NF-κB expression, and clinicopathological features.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques: Expressing, Adjuvant

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Association between PEDF, E-cadherin, vimentin, Snail and NF-κB expression in invasive breast carcinoma samples.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Correlation of PEDF, E-cadherin, vimentin, Snail and NF-κB expression with patient survival.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Multivariate analysis of overall survival of patients with invasive breast carcinoma.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques:

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Multivariate analysis of disease-free survival of patients with invasive breast carcinoma.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques:

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Kaplan-Meier analysis for overall survival curves of breast cancer patients with pigment epithelium-derived factor (PEDF), E-cadherin, vimentin, Snail and nuclear factor κB (NF-κB) expression. Survival curves are stratified by negative (−) and positive (+) (A) PEDF, (B) E-cadherin, (C) vimentin, (D) Snail and (E) NF-κB expression.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques: Derivative Assay, Expressing

Journal: Oncology Letters

Article Title: Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma

doi: 10.3892/ol.2015.3880

Figure Lengend Snippet: Kaplan-Meier analysis for disease-free survival curves of breast cancer patients with pigment epithelium-derived factor (PEDF), E-cadherin, vimentin, Snail and nuclear factor κB (NF-κB) expression. Survival curves are stratified by negative (−) and positive (+) (A) PEDF, (B) E-cadherin, (C) vimentin, (D) Snail and (E) NF-κB expression.

Article Snippet: Subsequent to blocking endogenous peroxidase (3% hydrogen peroxidase; Beyotime Biotechnology), the sections were incubated with primary mouse anti-human monoclonal PEDF (1:100; Millipore, Billerica, MA, USA), rabbit anti-human monoclonal E-cadherin (1:500; Millipore), mouse anti-human monoclonal vimentin (1:100; Cell Signaling Technology Inc., Danvers, MA, USA), goat anti-human polyclonal Snail (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-human monoclonal NF-κB (1:600; Cell Signaling Technology Inc.) antibodies diluted in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and 5% bovine serum albumin (Beyotime Biotechnology) overnight at 4°C.

Techniques: Derivative Assay, Expressing

Journal:

Article Title: The alternative pathway is required, but not alone sufficient, for retinal pathology in mouse laser-induced choroidal neovascularization

doi: 10.1016/j.molimm.2010.12.016

Figure Lengend Snippet: Quantitative RT-PCR was performed on RPE-choroid samples removed 7 days post laser photocoagulation from control (C57BL/6), CFB−/− (no-AP) and C1q−/− MBL−/− (AP-only) mice. Quantitative values were obtained by cycle number (Ct value), determining the difference between the mean experimental (e.g., VEGF) and control (β-actin) ΔCt values. With the exception of C3 mRNA expression, mRNA levels for all the angiogenesis (VEGF and PEDF) and complement markers (C9) were normalized in CFB−/− (no-AP) and C1q−/− MBL−/− (AP-only) mice when compared to the C57BL/6 control. No consistent trends were obtained in the membrane-bound complement inhibitors CD55 and CD659 or the soluble inhibitor CFH. Tissues from 3 animals per group were evaluated.

Article Snippet: ELISA for VEGF and PEDF measurement To measure production of VEGF and PEDF by the RPE-choroid, eyecups were solubilized in CellLytic MT (mammalian tissue lysis/extraction reagent; Sigma) and centrifuged at 20,000 g for 5 min. Microplates were coated with either the anti-mouse VEGF (Antigenix America, Inc.) ( Rohrer et al., 2009 ) or the anti-human PEDF capture antibody (R&D Systems, Minneapolis, MN) ( Ma et al., 2009 ), and 100 μL of the tissue extract was added.

Techniques: Quantitative RT-PCR, Control, Expressing, Membrane

Journal:

Article Title: The alternative pathway is required, but not alone sufficient, for retinal pathology in mouse laser-induced choroidal neovascularization

doi: 10.1016/j.molimm.2010.12.016

Figure Lengend Snippet: Quantitative ELISA assays were performed on RPE-choroid samples removed 7 days post laser photocoagulation from control (C57BL/6), CFB−/− (no-AP) and C1q−/− MBL−/− (AP-only) mice. Quantitative values were obtained by using a standard curve of purified mouse protein (VEGF) or by determining the fold difference between experimental (e.g., CFB−/− and control C57BL/6) values (PEDF). Protein levels for VEGF (A) and PEDF (B) followed the mRNA levels; both were normalized in CFB−/− (no-AP) and C1q−/− MBL−/− (AP-only) mice when compared to the C57BL/6 control. Tissues from 3 animals per group were evaluated.

Article Snippet: ELISA for VEGF and PEDF measurement To measure production of VEGF and PEDF by the RPE-choroid, eyecups were solubilized in CellLytic MT (mammalian tissue lysis/extraction reagent; Sigma) and centrifuged at 20,000 g for 5 min. Microplates were coated with either the anti-mouse VEGF (Antigenix America, Inc.) ( Rohrer et al., 2009 ) or the anti-human PEDF capture antibody (R&D Systems, Minneapolis, MN) ( Ma et al., 2009 ), and 100 μL of the tissue extract was added.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Purification

Journal: Journal of Cellular and Molecular Medicine

Article Title: The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

doi: 10.1111/j.1582-4934.2009.00731.x

Figure Lengend Snippet: PEDF expression in human cardiac tissue. PEDF and HIF-1α determination by Western blot of homogenized cardiac tissue isolated from two healthy hearts (lanes 1 and 2) and four explanted hearts from patients suffering from ischemic (lanes 3 and 4) and dilatative (lanes 5 and 6) cardiomyopathy (A). PEDF mRNA expression in human cardiac tissue isolated from the same hearts used for Western blot in (A), e.g. two healthy hearts (lanes 1 and 2) and four explanted hearts from patients suffering from ischemic (lanes 3 and 4) and dilatative (lanes 5 and 6) cardiomyopathy; GAPDH served as a loading control (B). Immunohistochemical staining of PEDF, troponin I and actin in paraffin embedded heart tissue from a healthy heart and explanted hearts from patients suffering from ischemic and dilatative cardiomyopathy, respectively (C). mRNA was isolated from the left ventricle of healthy human hearts ( n = 4) and from hearts of patients suffering from ischemic ( n = 8; * P = 0.014) or dilatative cardiomyopathy ( n = 17; n.s., P = 0.287); real-time PCR was performed employing specific primers for PEDF. Values represent mean values ± S.D. Values are given as x -fold of control and were normalized using GAPDH levels (D).

Article Snippet: We used an anti-human PEDF goat antibody (15 μg/ml; R&D Systems) for PEDF staining, an anti-human cardiac troponin I rabbit monoclonal antibody (1/250 dilution; Abcam, Cambridge, UK) for cardiac muscle staining and an anti-human smooth muscle α-actin mouse monoclonal antibody (ready to use, BioGenex).

Techniques: Expressing, Western Blot, Isolation, Control, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction