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Danaher Inc anti mouse rabbit polyclonal s100a4
Anti Mouse Rabbit Polyclonal S100a4, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Agilent technologies rabbit polyclonal s100a4
Abundance of total <t>S100A4</t> in a) the arterial wall in small, medium and large arteries; b) different arterial layers of small arteries (100–299 µm); c) different arterial layers of medium arteries (300–499 µm); and d) different arterial layers of large arteries (500–999 µm). NC: nonsmoker controls; NLFS: normal lung function smokers; SAD: patients with small airway disease; COPD-CS: current smokers with COPD; COPD-ES: ex-smokers with COPD. *: p≤0.05; **: p≤0.01.
Rabbit Polyclonal S100a4, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Abcam rabbit polyclonal fsp1 s100a4
a-b, UMAP (a) and cell state assignment (b) of skin fibroblasts from naked mole-rat (Hg), human (Hs) and mouse (Mm), sorted by condition: untreated (UN), DMBA/TPA (DT) treated, and papilloma (Pap.). HF: Hair follicle. Arrows: significantly (P < 0.05) increased (black) and decreased (grey) cell proportions. c, Predicted location of fibroblasts in the upper or lower dermis before and after DT treatment in Hg (class 1-6) and Mm (class 7-15). d-e , Transcriptional signatures in fibroblasts in class 1-6 (d) identify significantly (P = 0.0013) (e) enhanced migration in the upper dermis following DT treatment. f-g , Immunofluorescence of Fibroblast-specific protein 1 <t>(FSP1)</t> and keratin 14 (KRT14) (f) and number of cells in the upper dermis (g) in UN and DT treated skin of Mm and Hg. Dotted line: Basement membrane. Double arrow: Upper dermis (40 μm). DAPI: nuclear counterstain. Scale bar: 10 μm. h - i , Illustration of cell-cell interactions (h) and changes in cell-cell communications (i) between fibroblasts of class 1-6 following DT treatment shown as log ratio (log Δ) of percentage of significant interactions in DT over percentage of significant interactions in UN. j , Gene ontology analysis (GOrilla; cellular component) using significantly differently (adjusted P < 0.05) expressed genes in all Hg fibroblasts combined. Background: All expressed genes. Extracell.: Extracellular. k - l , Natural log transformed (Log) ratio of gene sets scores for collagen biosynthesis (k) and immune activation (l) comparing DT (filled circle) or Pap. (non-filled circle) over UN for indicated fibroblast subtype. m , Illustration (top) and Log ratio (bottom) of myofibroblasts over pro-inflammatory cancer associated fibroblasts (myCAF/iCAF) gene sets scores in DT (black circle) and Pap. (white circle) fibroblast subtype 11-13 and 15. n , Percent change in UN and DT cell-cell integrin interactions across all epithelial cells (Epi.) and fibroblasts (Fib.) in Hg and Mm skin. Box plots show first quartile, median, and third quartile (g) or median and whiskers at 5 and 95 percentiles (n). *: P < 0.05 (k-l). Unpaired two-tailed unpaired t -test (g). F-test (n). P value for interaction term between dermal location and treatment in a linear mixed-effects model describing fibroblast migration score (e) and myCAF/iCAF ratio (m).
Rabbit Polyclonal Fsp1 S100a4, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Danaher Inc rabbit polyclonal anti s100a4
a-b, UMAP (a) and cell state assignment (b) of skin fibroblasts from naked mole-rat (Hg), human (Hs) and mouse (Mm), sorted by condition: untreated (UN), DMBA/TPA (DT) treated, and papilloma (Pap.). HF: Hair follicle. Arrows: significantly (P < 0.05) increased (black) and decreased (grey) cell proportions. c, Predicted location of fibroblasts in the upper or lower dermis before and after DT treatment in Hg (class 1-6) and Mm (class 7-15). d-e , Transcriptional signatures in fibroblasts in class 1-6 (d) identify significantly (P = 0.0013) (e) enhanced migration in the upper dermis following DT treatment. f-g , Immunofluorescence of Fibroblast-specific protein 1 <t>(FSP1)</t> and keratin 14 (KRT14) (f) and number of cells in the upper dermis (g) in UN and DT treated skin of Mm and Hg. Dotted line: Basement membrane. Double arrow: Upper dermis (40 μm). DAPI: nuclear counterstain. Scale bar: 10 μm. h - i , Illustration of cell-cell interactions (h) and changes in cell-cell communications (i) between fibroblasts of class 1-6 following DT treatment shown as log ratio (log Δ) of percentage of significant interactions in DT over percentage of significant interactions in UN. j , Gene ontology analysis (GOrilla; cellular component) using significantly differently (adjusted P < 0.05) expressed genes in all Hg fibroblasts combined. Background: All expressed genes. Extracell.: Extracellular. k - l , Natural log transformed (Log) ratio of gene sets scores for collagen biosynthesis (k) and immune activation (l) comparing DT (filled circle) or Pap. (non-filled circle) over UN for indicated fibroblast subtype. m , Illustration (top) and Log ratio (bottom) of myofibroblasts over pro-inflammatory cancer associated fibroblasts (myCAF/iCAF) gene sets scores in DT (black circle) and Pap. (white circle) fibroblast subtype 11-13 and 15. n , Percent change in UN and DT cell-cell integrin interactions across all epithelial cells (Epi.) and fibroblasts (Fib.) in Hg and Mm skin. Box plots show first quartile, median, and third quartile (g) or median and whiskers at 5 and 95 percentiles (n). *: P < 0.05 (k-l). Unpaired two-tailed unpaired t -test (g). F-test (n). P value for interaction term between dermal location and treatment in a linear mixed-effects model describing fibroblast migration score (e) and myCAF/iCAF ratio (m).
Rabbit Polyclonal Anti S100a4, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Thermo Fisher polyclonal rabbit anti s100a4
<t>S100A4</t> mRNA expression is an unfavorable prognostic factor for colon and rectal cancer patients. (A) Kaplan–Meier curve of TCGA survival data (OS, DSS, DFS, and PFS) for high-risk and low-risk patients in combined CRC group. (B) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups in colon cancer patients. (C) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups of rectal cancer patients. Log-rank test p-values are shown in Kaplan–Meier curves. (D) S100A4 mRNA expression is associated with vascular invasion in rectal cancer patients (TCGA-READ data). Mann-Whitney U test was applied. Box plot depicts gene expression (min, Q1, median, Q3, max).
Polyclonal Rabbit Anti S100a4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology rabbit polyclonal anti s100a4
<t>S100A4</t> mRNA expression is an unfavorable prognostic factor for colon and rectal cancer patients. (A) Kaplan–Meier curve of TCGA survival data (OS, DSS, DFS, and PFS) for high-risk and low-risk patients in combined CRC group. (B) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups in colon cancer patients. (C) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups of rectal cancer patients. Log-rank test p-values are shown in Kaplan–Meier curves. (D) S100A4 mRNA expression is associated with vascular invasion in rectal cancer patients (TCGA-READ data). Mann-Whitney U test was applied. Box plot depicts gene expression (min, Q1, median, Q3, max).
Rabbit Polyclonal Anti S100a4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Agilent technologies polyclonal rabbit anti human s100a4 antibody
Effect of cantharidin and norcantharidin on <t>S100A4</t> mRNA and protein expression after 24 h and 48 h. The HCT116 and SW620 cells were treated daily with increasing concentrations (0.1–30 µM) vs. the DMSO control. RNA was extracted and RT-qPCR was conducted for the S100A4 mRNA represented as the percentage of the DMSO control of three independent experiments. The black bar indicates the DMSO control and the grey bars are the experimental samples which represent the data mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001. Protein levels were analyzed with an immunoblot assay with β-actin as a loading control. The blots represent one of the three independent experiments. ( A ) Effect of cantharidin on S100A4 mRNA and protein expression in HCT116 and SW620 cells. Top : 24 h treatment and bottom : 48 h treatment. HCT116 cells left side, SW620 cells right side. ( B ) Effect of norcantharidin on S100A4 mRNA and protein expression in HCT116 and SW620 cells. Top : 24 h treatment and bottom : 48 h treatment. HCT116 cells on the left side and SW620 cells on the right side.
Polyclonal Rabbit Anti Human S100a4 Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/polyclonal rabbit anti human s100a4 antibody/product/Agilent technologies
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Thermo Fisher rabbit polyclonal anti human s100a4 antibody
Primer sequences and amplicon sizes. The primer sets work under identical PCR cycling conditions to obtain simultaneous amplification in the same run. Sequences were taken from GeneBank, Accession numbers are given.
Rabbit Polyclonal Anti Human S100a4 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Agilent technologies rabbit polyclonal anti s100a4 secondary antibody
A) Dose-response of VEGF and <t>S100A4.</t> Cells were treated with EBM, S100A4, VEGF, or the combination of S100A4 plus VEGF for 24 h and migration was analyzed. B) Western-blot analysis of KDR expression after adding either S100A4 (0.3–30 µM) or VEGF (1–100 ng/mL) for 24 h. C) Levels of KDR expression and activation (phosphorylation) induced by VEGF (10 ng/mL), S100A4 (3 µM) or by the combination of the two proteins for 24 h of stimulation. All KDR signal intensities were normalized to α-tubulin. D) Time course (4 h and 8 h) of KDR expression and KDR phosphorylation upon incubation with S100A4 (3 µM) or VEGF (10 ng/mL) or the combination of the two proteins. E) Proteolytic activity of MMPs in HUVEC conditioned media. Cells were treated with S100A4 in EBM alone for 24 h. Active forms of MMP-9 induced by S100A4 are indicated by arrowheads. F) HUVEC were treated with VEGF (3 ng/mL), VEGF plus S100A4 (1 µM) or the combination of these proteins with the antibody 5C3 (0.25–4 µM) for 24 h. 5H4 was used as non-blocking antibody. G) 5C3 (1–2 µM) neutralized the production of active forms of MMP-9 induced by S100A4 (arrowhead). Each data point was normalized to the positive control of cells incubated with complete medium (left bar) that represents 100% migration. The control is the maximum of the possible migration (migration control). This migration is obtained by incubating HUVEC with basal medium containing 10% FCS plus hydrocortisone, brain bovine extract and hEGF. All the experimental points are referred to this control. Bars show the mean ± SEM. ns p >0.05, * p <0.05, **p<0.01, *** p <0.001.
Rabbit Polyclonal Anti S100a4 Secondary Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Abundance of total S100A4 in a) the arterial wall in small, medium and large arteries; b) different arterial layers of small arteries (100–299 µm); c) different arterial layers of medium arteries (300–499 µm); and d) different arterial layers of large arteries (500–999 µm). NC: nonsmoker controls; NLFS: normal lung function smokers; SAD: patients with small airway disease; COPD-CS: current smokers with COPD; COPD-ES: ex-smokers with COPD. *: p≤0.05; **: p≤0.01.

Journal: ERJ Open Research

Article Title: Endothelial to mesenchymal transition is an active process in smokers and patients with early COPD contributing to pulmonary arterial pathology

doi: 10.1183/23120541.00767-2023

Figure Lengend Snippet: Abundance of total S100A4 in a) the arterial wall in small, medium and large arteries; b) different arterial layers of small arteries (100–299 µm); c) different arterial layers of medium arteries (300–499 µm); and d) different arterial layers of large arteries (500–999 µm). NC: nonsmoker controls; NLFS: normal lung function smokers; SAD: patients with small airway disease; COPD-CS: current smokers with COPD; COPD-ES: ex-smokers with COPD. *: p≤0.05; **: p≤0.01.

Article Snippet: The tissues were stained with EndMT primary antibodies, monoclonal mouse anti-human CD31 (1:40; Dako M0823, Agilent Technologies), rabbit polyclonal S100A4 (1:1000; Dako A5114, Agilent Technologies), mouse monoclonal vimentin (1:200; Dako M7020, Agilent Technologies) and mouse monoclonal anti-N-cadherin (1:100; Ab98952, Abcam, Melbourne, VIC, Australia) for 60 min at ambient temperature, followed by secondary horseradish peroxidase rabbit/mouse antibodies (EnVision Detection System, Dako K5007, Agilent Technologies) for 30 min.

Techniques:

Correlation between a) total vimentin and forced expiratory volume in 1 s (FEV 1 )/forced vital capacity (FVC), b) total vimentin and forced expiratory fraction at 25–75% of FVC (FEF 25–75% ), c) intimal S100A4 and FEV 1 /FVC and d) intimal S100A4 and FEF 25–75% across arterial sizes.

Journal: ERJ Open Research

Article Title: Endothelial to mesenchymal transition is an active process in smokers and patients with early COPD contributing to pulmonary arterial pathology

doi: 10.1183/23120541.00767-2023

Figure Lengend Snippet: Correlation between a) total vimentin and forced expiratory volume in 1 s (FEV 1 )/forced vital capacity (FVC), b) total vimentin and forced expiratory fraction at 25–75% of FVC (FEF 25–75% ), c) intimal S100A4 and FEV 1 /FVC and d) intimal S100A4 and FEF 25–75% across arterial sizes.

Article Snippet: The tissues were stained with EndMT primary antibodies, monoclonal mouse anti-human CD31 (1:40; Dako M0823, Agilent Technologies), rabbit polyclonal S100A4 (1:1000; Dako A5114, Agilent Technologies), mouse monoclonal vimentin (1:200; Dako M7020, Agilent Technologies) and mouse monoclonal anti-N-cadherin (1:100; Ab98952, Abcam, Melbourne, VIC, Australia) for 60 min at ambient temperature, followed by secondary horseradish peroxidase rabbit/mouse antibodies (EnVision Detection System, Dako K5007, Agilent Technologies) for 30 min.

Techniques:

Correlation between a) cytoplasmic CD31 + cells (%) and diffusing capacity of the lung for carbon monoxide ( D LCO ), b) intimal vimentin abundance and D LCO , c) total S100A4 abundance and total arterial wall thickness and d) cytoplasmic CD31 + cells (%) and intimal thickness across small (100–299 µm), medium (300–499 µm) and large (500–999 µm) arteries.

Journal: ERJ Open Research

Article Title: Endothelial to mesenchymal transition is an active process in smokers and patients with early COPD contributing to pulmonary arterial pathology

doi: 10.1183/23120541.00767-2023

Figure Lengend Snippet: Correlation between a) cytoplasmic CD31 + cells (%) and diffusing capacity of the lung for carbon monoxide ( D LCO ), b) intimal vimentin abundance and D LCO , c) total S100A4 abundance and total arterial wall thickness and d) cytoplasmic CD31 + cells (%) and intimal thickness across small (100–299 µm), medium (300–499 µm) and large (500–999 µm) arteries.

Article Snippet: The tissues were stained with EndMT primary antibodies, monoclonal mouse anti-human CD31 (1:40; Dako M0823, Agilent Technologies), rabbit polyclonal S100A4 (1:1000; Dako A5114, Agilent Technologies), mouse monoclonal vimentin (1:200; Dako M7020, Agilent Technologies) and mouse monoclonal anti-N-cadherin (1:100; Ab98952, Abcam, Melbourne, VIC, Australia) for 60 min at ambient temperature, followed by secondary horseradish peroxidase rabbit/mouse antibodies (EnVision Detection System, Dako K5007, Agilent Technologies) for 30 min.

Techniques:

a-b, UMAP (a) and cell state assignment (b) of skin fibroblasts from naked mole-rat (Hg), human (Hs) and mouse (Mm), sorted by condition: untreated (UN), DMBA/TPA (DT) treated, and papilloma (Pap.). HF: Hair follicle. Arrows: significantly (P < 0.05) increased (black) and decreased (grey) cell proportions. c, Predicted location of fibroblasts in the upper or lower dermis before and after DT treatment in Hg (class 1-6) and Mm (class 7-15). d-e , Transcriptional signatures in fibroblasts in class 1-6 (d) identify significantly (P = 0.0013) (e) enhanced migration in the upper dermis following DT treatment. f-g , Immunofluorescence of Fibroblast-specific protein 1 (FSP1) and keratin 14 (KRT14) (f) and number of cells in the upper dermis (g) in UN and DT treated skin of Mm and Hg. Dotted line: Basement membrane. Double arrow: Upper dermis (40 μm). DAPI: nuclear counterstain. Scale bar: 10 μm. h - i , Illustration of cell-cell interactions (h) and changes in cell-cell communications (i) between fibroblasts of class 1-6 following DT treatment shown as log ratio (log Δ) of percentage of significant interactions in DT over percentage of significant interactions in UN. j , Gene ontology analysis (GOrilla; cellular component) using significantly differently (adjusted P < 0.05) expressed genes in all Hg fibroblasts combined. Background: All expressed genes. Extracell.: Extracellular. k - l , Natural log transformed (Log) ratio of gene sets scores for collagen biosynthesis (k) and immune activation (l) comparing DT (filled circle) or Pap. (non-filled circle) over UN for indicated fibroblast subtype. m , Illustration (top) and Log ratio (bottom) of myofibroblasts over pro-inflammatory cancer associated fibroblasts (myCAF/iCAF) gene sets scores in DT (black circle) and Pap. (white circle) fibroblast subtype 11-13 and 15. n , Percent change in UN and DT cell-cell integrin interactions across all epithelial cells (Epi.) and fibroblasts (Fib.) in Hg and Mm skin. Box plots show first quartile, median, and third quartile (g) or median and whiskers at 5 and 95 percentiles (n). *: P < 0.05 (k-l). Unpaired two-tailed unpaired t -test (g). F-test (n). P value for interaction term between dermal location and treatment in a linear mixed-effects model describing fibroblast migration score (e) and myCAF/iCAF ratio (m).

Journal: bioRxiv

Article Title: An interactive cellular ecosystem blocks epithelial transformation in naked mole-rat

doi: 10.1101/2023.10.10.561212

Figure Lengend Snippet: a-b, UMAP (a) and cell state assignment (b) of skin fibroblasts from naked mole-rat (Hg), human (Hs) and mouse (Mm), sorted by condition: untreated (UN), DMBA/TPA (DT) treated, and papilloma (Pap.). HF: Hair follicle. Arrows: significantly (P < 0.05) increased (black) and decreased (grey) cell proportions. c, Predicted location of fibroblasts in the upper or lower dermis before and after DT treatment in Hg (class 1-6) and Mm (class 7-15). d-e , Transcriptional signatures in fibroblasts in class 1-6 (d) identify significantly (P = 0.0013) (e) enhanced migration in the upper dermis following DT treatment. f-g , Immunofluorescence of Fibroblast-specific protein 1 (FSP1) and keratin 14 (KRT14) (f) and number of cells in the upper dermis (g) in UN and DT treated skin of Mm and Hg. Dotted line: Basement membrane. Double arrow: Upper dermis (40 μm). DAPI: nuclear counterstain. Scale bar: 10 μm. h - i , Illustration of cell-cell interactions (h) and changes in cell-cell communications (i) between fibroblasts of class 1-6 following DT treatment shown as log ratio (log Δ) of percentage of significant interactions in DT over percentage of significant interactions in UN. j , Gene ontology analysis (GOrilla; cellular component) using significantly differently (adjusted P < 0.05) expressed genes in all Hg fibroblasts combined. Background: All expressed genes. Extracell.: Extracellular. k - l , Natural log transformed (Log) ratio of gene sets scores for collagen biosynthesis (k) and immune activation (l) comparing DT (filled circle) or Pap. (non-filled circle) over UN for indicated fibroblast subtype. m , Illustration (top) and Log ratio (bottom) of myofibroblasts over pro-inflammatory cancer associated fibroblasts (myCAF/iCAF) gene sets scores in DT (black circle) and Pap. (white circle) fibroblast subtype 11-13 and 15. n , Percent change in UN and DT cell-cell integrin interactions across all epithelial cells (Epi.) and fibroblasts (Fib.) in Hg and Mm skin. Box plots show first quartile, median, and third quartile (g) or median and whiskers at 5 and 95 percentiles (n). *: P < 0.05 (k-l). Unpaired two-tailed unpaired t -test (g). F-test (n). P value for interaction term between dermal location and treatment in a linear mixed-effects model describing fibroblast migration score (e) and myCAF/iCAF ratio (m).

Article Snippet: Primary antibodies were diluted in 1% FBS and incubated overnight using the following dilutions: mouse monoclonal KRT14 (1:1000, Abcam, ab7800), rabbit monoclonal KRT10 (1:200, Abcam, ab76318), mouse monoclonal to KRT6 (1:200, Abcam, ab18586), rabbit polyclonal CD3 (1:200, Abcam, ab5690), rabbit polyclonal FSP1/S100A4 (1:100, Abcam, ab41532), rabbit polyclonal Ki67/MKI67 (1:200, Abcam, ab15580).

Techniques: Migration, Immunofluorescence, Transformation Assay, Activation Assay, Two Tailed Test

S100A4 mRNA expression is an unfavorable prognostic factor for colon and rectal cancer patients. (A) Kaplan–Meier curve of TCGA survival data (OS, DSS, DFS, and PFS) for high-risk and low-risk patients in combined CRC group. (B) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups in colon cancer patients. (C) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups of rectal cancer patients. Log-rank test p-values are shown in Kaplan–Meier curves. (D) S100A4 mRNA expression is associated with vascular invasion in rectal cancer patients (TCGA-READ data). Mann-Whitney U test was applied. Box plot depicts gene expression (min, Q1, median, Q3, max).

Journal: Frontiers in Oncology

Article Title: Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers

doi: 10.3389/fonc.2023.1058337

Figure Lengend Snippet: S100A4 mRNA expression is an unfavorable prognostic factor for colon and rectal cancer patients. (A) Kaplan–Meier curve of TCGA survival data (OS, DSS, DFS, and PFS) for high-risk and low-risk patients in combined CRC group. (B) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups in colon cancer patients. (C) Kaplan–Meier curve of TCGA survival data for high-risk and low-risk groups of rectal cancer patients. Log-rank test p-values are shown in Kaplan–Meier curves. (D) S100A4 mRNA expression is associated with vascular invasion in rectal cancer patients (TCGA-READ data). Mann-Whitney U test was applied. Box plot depicts gene expression (min, Q1, median, Q3, max).

Article Snippet: For IF double staining mouse anti-CD68 monoclonal antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); polyclonal rabbit anti-S100A4 (1:1000, PA5-82322, Thermo Fisher Scientific, USA), polyclonal goat anti-SPARC (1:80, AF941, R&D Systems, USA) and polyclonal goat anti-SPP1 (1:80, AF1433, R&D Systems, USA) were used.

Techniques: Expressing, MANN-WHITNEY

The prognostic significance of  S100A4  mRNA levels for disease-free survival in patients with CRC revealed by univariate and multivariate COX analysis.

Journal: Frontiers in Oncology

Article Title: Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers

doi: 10.3389/fonc.2023.1058337

Figure Lengend Snippet: The prognostic significance of S100A4 mRNA levels for disease-free survival in patients with CRC revealed by univariate and multivariate COX analysis.

Article Snippet: For IF double staining mouse anti-CD68 monoclonal antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); polyclonal rabbit anti-S100A4 (1:1000, PA5-82322, Thermo Fisher Scientific, USA), polyclonal goat anti-SPARC (1:80, AF941, R&D Systems, USA) and polyclonal goat anti-SPP1 (1:80, AF1433, R&D Systems, USA) were used.

Techniques: Expressing

The prognostic significance of  S100A4  mRNA levels for disease-free survival with colon cancer revealed by univariate and multivariate COX analysis.

Journal: Frontiers in Oncology

Article Title: Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers

doi: 10.3389/fonc.2023.1058337

Figure Lengend Snippet: The prognostic significance of S100A4 mRNA levels for disease-free survival with colon cancer revealed by univariate and multivariate COX analysis.

Article Snippet: For IF double staining mouse anti-CD68 monoclonal antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); polyclonal rabbit anti-S100A4 (1:1000, PA5-82322, Thermo Fisher Scientific, USA), polyclonal goat anti-SPARC (1:80, AF941, R&D Systems, USA) and polyclonal goat anti-SPP1 (1:80, AF1433, R&D Systems, USA) were used.

Techniques: Expressing

S100A4, SPARC and SPP1 are expressed by tumor-associated macrophages in colorectal cancer. (A) IHC representative images (x400) of stromal S100A4, SPARC and SPP1 in tumor tissue. Zoom images is given in the lower panel (x800). Stromal expression of S100A4 and SPARC is higher than tumor expression. The protein level of SPP1 does not have differences between tumor and stroma. Student’s t-test was applied. (B) Immune cell-type deconvolution analysis that was performed via the TIMER2.0 web platform using the CIBERSORT method. S100A4, SPP1 and SPARC gene expression correlates with macrophages. *: |rho|>0.2 and FDR<0.05. (C) Nanostring GeoMX DSP revealed increased expression of SPP1 in the immune compartment and its correlation with macrophages. *: |rho|>0.2 and FDR<0.05. (D) Confocal microscopy confirmed the co-localization of S100A4, SPARC and SPP1 in CD68+ TAMs. Scale bars equal 20 µm.

Journal: Frontiers in Oncology

Article Title: Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers

doi: 10.3389/fonc.2023.1058337

Figure Lengend Snippet: S100A4, SPARC and SPP1 are expressed by tumor-associated macrophages in colorectal cancer. (A) IHC representative images (x400) of stromal S100A4, SPARC and SPP1 in tumor tissue. Zoom images is given in the lower panel (x800). Stromal expression of S100A4 and SPARC is higher than tumor expression. The protein level of SPP1 does not have differences between tumor and stroma. Student’s t-test was applied. (B) Immune cell-type deconvolution analysis that was performed via the TIMER2.0 web platform using the CIBERSORT method. S100A4, SPP1 and SPARC gene expression correlates with macrophages. *: |rho|>0.2 and FDR<0.05. (C) Nanostring GeoMX DSP revealed increased expression of SPP1 in the immune compartment and its correlation with macrophages. *: |rho|>0.2 and FDR<0.05. (D) Confocal microscopy confirmed the co-localization of S100A4, SPARC and SPP1 in CD68+ TAMs. Scale bars equal 20 µm.

Article Snippet: For IF double staining mouse anti-CD68 monoclonal antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); polyclonal rabbit anti-S100A4 (1:1000, PA5-82322, Thermo Fisher Scientific, USA), polyclonal goat anti-SPARC (1:80, AF941, R&D Systems, USA) and polyclonal goat anti-SPP1 (1:80, AF1433, R&D Systems, USA) were used.

Techniques: Expressing, Confocal Microscopy

The association of stromal expression of S100A4, SPARC and SPP1 with survival and clinical-pathological parameters. (A) S100A4 stromal expression predicts poor OS for CRC patients and colon cancer patients, but is negatively correlated with tumor stage in rectal cancer. (B) The prognostic significance of S100A4 protein levels for overall survival in patients with CRC and CC revealed by univariate and multivariate COX analysis. (C) SPARC stromal expression is an unfavorable parameter for RFS in rectal cancer patients. SPARC protein expression is negatively associated with clinical-pathological parameters in combined CRC group and in rectal cancer patients. (D) SPP1 stromal expression is unfavorable for PFS in patients with rectal cancer. SPP1 protein expression is negatively associated with clinical-pathological parameters in combined CRC group and in rectal cancer patients. Log-rank test p-values are shown in Kaplan-Meier plots. Mann-Whitney U test was applied for the comparison of two groups. Box plots depict protein expression (min, Q1, median, Q3, max).

Journal: Frontiers in Oncology

Article Title: Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers

doi: 10.3389/fonc.2023.1058337

Figure Lengend Snippet: The association of stromal expression of S100A4, SPARC and SPP1 with survival and clinical-pathological parameters. (A) S100A4 stromal expression predicts poor OS for CRC patients and colon cancer patients, but is negatively correlated with tumor stage in rectal cancer. (B) The prognostic significance of S100A4 protein levels for overall survival in patients with CRC and CC revealed by univariate and multivariate COX analysis. (C) SPARC stromal expression is an unfavorable parameter for RFS in rectal cancer patients. SPARC protein expression is negatively associated with clinical-pathological parameters in combined CRC group and in rectal cancer patients. (D) SPP1 stromal expression is unfavorable for PFS in patients with rectal cancer. SPP1 protein expression is negatively associated with clinical-pathological parameters in combined CRC group and in rectal cancer patients. Log-rank test p-values are shown in Kaplan-Meier plots. Mann-Whitney U test was applied for the comparison of two groups. Box plots depict protein expression (min, Q1, median, Q3, max).

Article Snippet: For IF double staining mouse anti-CD68 monoclonal antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); polyclonal rabbit anti-S100A4 (1:1000, PA5-82322, Thermo Fisher Scientific, USA), polyclonal goat anti-SPARC (1:80, AF941, R&D Systems, USA) and polyclonal goat anti-SPP1 (1:80, AF1433, R&D Systems, USA) were used.

Techniques: Expressing, MANN-WHITNEY

S100A4 is associated with improved outcomes in patients undergone neoadjuvant chemotherapy-based treatment. (A) Stromal expression of S100A4 decreased in treated patients and correlated with favorable response to NACT/CRT. (B) S100A4 and SPARC mRNA expression decreased in non-responders with progression. S100A4 high-risk score predicts better DFS. Mann-Whitney U test was applied. Box plots depict protein (A) and gene (B) expression (min, Q1, median, Q3, max). Log-rank test p-value is shown in Kaplan-Meier plot.

Journal: Frontiers in Oncology

Article Title: Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers

doi: 10.3389/fonc.2023.1058337

Figure Lengend Snippet: S100A4 is associated with improved outcomes in patients undergone neoadjuvant chemotherapy-based treatment. (A) Stromal expression of S100A4 decreased in treated patients and correlated with favorable response to NACT/CRT. (B) S100A4 and SPARC mRNA expression decreased in non-responders with progression. S100A4 high-risk score predicts better DFS. Mann-Whitney U test was applied. Box plots depict protein (A) and gene (B) expression (min, Q1, median, Q3, max). Log-rank test p-value is shown in Kaplan-Meier plot.

Article Snippet: For IF double staining mouse anti-CD68 monoclonal antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); polyclonal rabbit anti-S100A4 (1:1000, PA5-82322, Thermo Fisher Scientific, USA), polyclonal goat anti-SPARC (1:80, AF941, R&D Systems, USA) and polyclonal goat anti-SPP1 (1:80, AF1433, R&D Systems, USA) were used.

Techniques: Expressing, MANN-WHITNEY

Effect of cantharidin and norcantharidin on S100A4 mRNA and protein expression after 24 h and 48 h. The HCT116 and SW620 cells were treated daily with increasing concentrations (0.1–30 µM) vs. the DMSO control. RNA was extracted and RT-qPCR was conducted for the S100A4 mRNA represented as the percentage of the DMSO control of three independent experiments. The black bar indicates the DMSO control and the grey bars are the experimental samples which represent the data mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001. Protein levels were analyzed with an immunoblot assay with β-actin as a loading control. The blots represent one of the three independent experiments. ( A ) Effect of cantharidin on S100A4 mRNA and protein expression in HCT116 and SW620 cells. Top : 24 h treatment and bottom : 48 h treatment. HCT116 cells left side, SW620 cells right side. ( B ) Effect of norcantharidin on S100A4 mRNA and protein expression in HCT116 and SW620 cells. Top : 24 h treatment and bottom : 48 h treatment. HCT116 cells on the left side and SW620 cells on the right side.

Journal: International Journal of Molecular Sciences

Article Title: Cantharidin and Its Analogue Norcantharidin Inhibit Metastasis—Inducing Genes S100A4 and MACC1

doi: 10.3390/ijms24021179

Figure Lengend Snippet: Effect of cantharidin and norcantharidin on S100A4 mRNA and protein expression after 24 h and 48 h. The HCT116 and SW620 cells were treated daily with increasing concentrations (0.1–30 µM) vs. the DMSO control. RNA was extracted and RT-qPCR was conducted for the S100A4 mRNA represented as the percentage of the DMSO control of three independent experiments. The black bar indicates the DMSO control and the grey bars are the experimental samples which represent the data mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001. Protein levels were analyzed with an immunoblot assay with β-actin as a loading control. The blots represent one of the three independent experiments. ( A ) Effect of cantharidin on S100A4 mRNA and protein expression in HCT116 and SW620 cells. Top : 24 h treatment and bottom : 48 h treatment. HCT116 cells left side, SW620 cells right side. ( B ) Effect of norcantharidin on S100A4 mRNA and protein expression in HCT116 and SW620 cells. Top : 24 h treatment and bottom : 48 h treatment. HCT116 cells on the left side and SW620 cells on the right side.

Article Snippet: The polyclonal rabbit anti-human S100A4 antibody was purchased from Dako (Glostrup, Denmark) and the polyclonal anti-human MACC1 antibody was purchased from Sigma-Aldrich (Schnelldorf, Germany).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Time-dependent analysis of gene expression inhibition by cantharidin ( A ) and norcantharidin ( B ) of S100A4 ( top ) and MACC1 ( bottom ) in HCT116 ( left ) and SW620 ( right ) cells. The cells were treated once with 10 µM of cantharidin or norcantharidin and every 6 h the samples were taken over a time course of 60 h. RNA was extracted and RT-qPCR was conducted for S100A4, MACC1, and the control genes GAPDH and RPII. The black bar indicates the starting point at 0 h and the grey bars indicate each timepoint after 0h. The data represent mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001. ( A ) Time dependent inhibition of S100A4 gene ( top ) and MACC1 gene ( bottom ) expression after treatment of HCT116 ( left ) and SW620 cells ( right ) with 10 µM of cantharidin. ( B ) Time-dependent inhibition of S100A4 gene ( top ) and MACC1 gene ( bottom ) after treatment of HCT116 ( left ) and SW620 cells ( right ) with 10 µM of norcantharidin.

Journal: International Journal of Molecular Sciences

Article Title: Cantharidin and Its Analogue Norcantharidin Inhibit Metastasis—Inducing Genes S100A4 and MACC1

doi: 10.3390/ijms24021179

Figure Lengend Snippet: Time-dependent analysis of gene expression inhibition by cantharidin ( A ) and norcantharidin ( B ) of S100A4 ( top ) and MACC1 ( bottom ) in HCT116 ( left ) and SW620 ( right ) cells. The cells were treated once with 10 µM of cantharidin or norcantharidin and every 6 h the samples were taken over a time course of 60 h. RNA was extracted and RT-qPCR was conducted for S100A4, MACC1, and the control genes GAPDH and RPII. The black bar indicates the starting point at 0 h and the grey bars indicate each timepoint after 0h. The data represent mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001. ( A ) Time dependent inhibition of S100A4 gene ( top ) and MACC1 gene ( bottom ) expression after treatment of HCT116 ( left ) and SW620 cells ( right ) with 10 µM of cantharidin. ( B ) Time-dependent inhibition of S100A4 gene ( top ) and MACC1 gene ( bottom ) after treatment of HCT116 ( left ) and SW620 cells ( right ) with 10 µM of norcantharidin.

Article Snippet: The polyclonal rabbit anti-human S100A4 antibody was purchased from Dako (Glostrup, Denmark) and the polyclonal anti-human MACC1 antibody was purchased from Sigma-Aldrich (Schnelldorf, Germany).

Techniques: Expressing, Inhibition, Quantitative RT-PCR

Effect of cantharidin and norcantharidin on anchorage-independent colony formation and cell migration. ( A ) Anchorage-independent colony formation. The cells were plated as single cells in 0.33% (wt/vol) agarose and treatment was performed with solvent or 10 µM cantharidin or a norcantharidin-containing medium. After 7 days, colonies were visualized by light microscopy and counted for colony formation (>4 cells = 1 colony). Magnification = 10×. ( B ) Cell migration of cantharidin- or norcantharidin-treated cells (treatment as in ( A )). Cell migration was determined using Boyden chamber assay and expressed as percent of solvent-treated cells. ( C ) Effect of cantharidin on gene expression of S100A4 in HCT116 KO MACC1 cells. The means and 95% confidence intervals from the three independent experiments are presented in all of the panels. The data represent mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Cantharidin and Its Analogue Norcantharidin Inhibit Metastasis—Inducing Genes S100A4 and MACC1

doi: 10.3390/ijms24021179

Figure Lengend Snippet: Effect of cantharidin and norcantharidin on anchorage-independent colony formation and cell migration. ( A ) Anchorage-independent colony formation. The cells were plated as single cells in 0.33% (wt/vol) agarose and treatment was performed with solvent or 10 µM cantharidin or a norcantharidin-containing medium. After 7 days, colonies were visualized by light microscopy and counted for colony formation (>4 cells = 1 colony). Magnification = 10×. ( B ) Cell migration of cantharidin- or norcantharidin-treated cells (treatment as in ( A )). Cell migration was determined using Boyden chamber assay and expressed as percent of solvent-treated cells. ( C ) Effect of cantharidin on gene expression of S100A4 in HCT116 KO MACC1 cells. The means and 95% confidence intervals from the three independent experiments are presented in all of the panels. The data represent mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The polyclonal rabbit anti-human S100A4 antibody was purchased from Dako (Glostrup, Denmark) and the polyclonal anti-human MACC1 antibody was purchased from Sigma-Aldrich (Schnelldorf, Germany).

Techniques: Migration, Light Microscopy, Boyden Chamber Assay, Expressing

Primer sequences and amplicon sizes. The primer sets work under identical PCR cycling conditions to obtain simultaneous amplification in the same run. Sequences were taken from GeneBank, Accession numbers are given.

Journal: Respiratory Research

Article Title: Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

doi: 10.1186/1465-9921-6-109

Figure Lengend Snippet: Primer sequences and amplicon sizes. The primer sets work under identical PCR cycling conditions to obtain simultaneous amplification in the same run. Sequences were taken from GeneBank, Accession numbers are given.

Article Snippet: Following dilutions of primary antibodies were used: Rabbit polyclonal anti-human S100A4 antibody (Neomarkers, Fremont, CA) – 1:700, rabbit polyclonal anti-human FKBP1a antibody (Abcam, Cambridge, UK) – 1:300, rabbit polyclonal anti-human CD36 (Santa Cruz Biotech, California, USA) – 1:200.

Techniques: Amplification

Regulation of S100A4, CD36 and FKBP1a on mRNA and protein level. A) Comparison of regulation between laser-microdissected arteries and lung homogenate from 1, 7, and 21 days of hypoxia exposure. (Red: array; blue: TaqMan). B) Immunohistochemical staining of S100A4, CD36 and FKBP1a in the mouse lung.

Journal: Respiratory Research

Article Title: Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

doi: 10.1186/1465-9921-6-109

Figure Lengend Snippet: Regulation of S100A4, CD36 and FKBP1a on mRNA and protein level. A) Comparison of regulation between laser-microdissected arteries and lung homogenate from 1, 7, and 21 days of hypoxia exposure. (Red: array; blue: TaqMan). B) Immunohistochemical staining of S100A4, CD36 and FKBP1a in the mouse lung.

Article Snippet: Following dilutions of primary antibodies were used: Rabbit polyclonal anti-human S100A4 antibody (Neomarkers, Fremont, CA) – 1:700, rabbit polyclonal anti-human FKBP1a antibody (Abcam, Cambridge, UK) – 1:300, rabbit polyclonal anti-human CD36 (Santa Cruz Biotech, California, USA) – 1:200.

Techniques: Immunohistochemical staining, Staining

Immunolocalisation of S100A4. A) S100A4 protein (left panel) co-localises with alpha-smooth muscle actin (right panel). B) Small vessels (marked by arrows) are negative for S100A4 under normoxia (left panel) however stain positive for S100A4 after 21 days of hypoxia.

Journal: Respiratory Research

Article Title: Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

doi: 10.1186/1465-9921-6-109

Figure Lengend Snippet: Immunolocalisation of S100A4. A) S100A4 protein (left panel) co-localises with alpha-smooth muscle actin (right panel). B) Small vessels (marked by arrows) are negative for S100A4 under normoxia (left panel) however stain positive for S100A4 after 21 days of hypoxia.

Article Snippet: Following dilutions of primary antibodies were used: Rabbit polyclonal anti-human S100A4 antibody (Neomarkers, Fremont, CA) – 1:700, rabbit polyclonal anti-human FKBP1a antibody (Abcam, Cambridge, UK) – 1:300, rabbit polyclonal anti-human CD36 (Santa Cruz Biotech, California, USA) – 1:200.

Techniques: Staining

A) Dose-response of VEGF and S100A4. Cells were treated with EBM, S100A4, VEGF, or the combination of S100A4 plus VEGF for 24 h and migration was analyzed. B) Western-blot analysis of KDR expression after adding either S100A4 (0.3–30 µM) or VEGF (1–100 ng/mL) for 24 h. C) Levels of KDR expression and activation (phosphorylation) induced by VEGF (10 ng/mL), S100A4 (3 µM) or by the combination of the two proteins for 24 h of stimulation. All KDR signal intensities were normalized to α-tubulin. D) Time course (4 h and 8 h) of KDR expression and KDR phosphorylation upon incubation with S100A4 (3 µM) or VEGF (10 ng/mL) or the combination of the two proteins. E) Proteolytic activity of MMPs in HUVEC conditioned media. Cells were treated with S100A4 in EBM alone for 24 h. Active forms of MMP-9 induced by S100A4 are indicated by arrowheads. F) HUVEC were treated with VEGF (3 ng/mL), VEGF plus S100A4 (1 µM) or the combination of these proteins with the antibody 5C3 (0.25–4 µM) for 24 h. 5H4 was used as non-blocking antibody. G) 5C3 (1–2 µM) neutralized the production of active forms of MMP-9 induced by S100A4 (arrowhead). Each data point was normalized to the positive control of cells incubated with complete medium (left bar) that represents 100% migration. The control is the maximum of the possible migration (migration control). This migration is obtained by incubating HUVEC with basal medium containing 10% FCS plus hydrocortisone, brain bovine extract and hEGF. All the experimental points are referred to this control. Bars show the mean ± SEM. ns p >0.05, * p <0.05, **p<0.01, *** p <0.001.

Journal: PLoS ONE

Article Title: Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

doi: 10.1371/journal.pone.0072480

Figure Lengend Snippet: A) Dose-response of VEGF and S100A4. Cells were treated with EBM, S100A4, VEGF, or the combination of S100A4 plus VEGF for 24 h and migration was analyzed. B) Western-blot analysis of KDR expression after adding either S100A4 (0.3–30 µM) or VEGF (1–100 ng/mL) for 24 h. C) Levels of KDR expression and activation (phosphorylation) induced by VEGF (10 ng/mL), S100A4 (3 µM) or by the combination of the two proteins for 24 h of stimulation. All KDR signal intensities were normalized to α-tubulin. D) Time course (4 h and 8 h) of KDR expression and KDR phosphorylation upon incubation with S100A4 (3 µM) or VEGF (10 ng/mL) or the combination of the two proteins. E) Proteolytic activity of MMPs in HUVEC conditioned media. Cells were treated with S100A4 in EBM alone for 24 h. Active forms of MMP-9 induced by S100A4 are indicated by arrowheads. F) HUVEC were treated with VEGF (3 ng/mL), VEGF plus S100A4 (1 µM) or the combination of these proteins with the antibody 5C3 (0.25–4 µM) for 24 h. 5H4 was used as non-blocking antibody. G) 5C3 (1–2 µM) neutralized the production of active forms of MMP-9 induced by S100A4 (arrowhead). Each data point was normalized to the positive control of cells incubated with complete medium (left bar) that represents 100% migration. The control is the maximum of the possible migration (migration control). This migration is obtained by incubating HUVEC with basal medium containing 10% FCS plus hydrocortisone, brain bovine extract and hEGF. All the experimental points are referred to this control. Bars show the mean ± SEM. ns p >0.05, * p <0.05, **p<0.01, *** p <0.001.

Article Snippet: Dishes were washed eight times with washing buffer (PBS-0.1% Tween-20) and Rabbit polyclonal anti-S100A4 secondary antibody (Dako) at 4 µg/mL was added to the wells which were incubated for 1 h at 37°C.

Techniques: Migration, Western Blot, Expressing, Activation Assay, Incubation, Activity Assay, Blocking Assay, Positive Control

A) Dose-response activity of anti-RAGE (0.02–200 nM) on HUVEC migration. After starvation, cells were incubated with an anti-RAGE mAb for 2 h and then treated with VEGF (10 ng/mL) or VEGF plus S100A4 (3 µM), respectively, for 24 h. B) Design of homologous peptides to the extracellular domains of RAGE. C) Before migration, peptides were incubated with S100A4 for 2 h at 37°C, then HUVEC were treated with VEGF (10 ng/mL) or VEGF plus S100A4 (3 µM) with 30 µM of each peptide respectively for 24 h. Each data point was normalized to the positive control (left bar) that represents 100% migration. Bars show the mean ± SEM. ns p >0.05, * p <0.05, *** p <0.001.

Journal: PLoS ONE

Article Title: Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

doi: 10.1371/journal.pone.0072480

Figure Lengend Snippet: A) Dose-response activity of anti-RAGE (0.02–200 nM) on HUVEC migration. After starvation, cells were incubated with an anti-RAGE mAb for 2 h and then treated with VEGF (10 ng/mL) or VEGF plus S100A4 (3 µM), respectively, for 24 h. B) Design of homologous peptides to the extracellular domains of RAGE. C) Before migration, peptides were incubated with S100A4 for 2 h at 37°C, then HUVEC were treated with VEGF (10 ng/mL) or VEGF plus S100A4 (3 µM) with 30 µM of each peptide respectively for 24 h. Each data point was normalized to the positive control (left bar) that represents 100% migration. Bars show the mean ± SEM. ns p >0.05, * p <0.05, *** p <0.001.

Article Snippet: Dishes were washed eight times with washing buffer (PBS-0.1% Tween-20) and Rabbit polyclonal anti-S100A4 secondary antibody (Dako) at 4 µg/mL was added to the wells which were incubated for 1 h at 37°C.

Techniques: Activity Assay, Migration, Incubation, Positive Control

Cells were treated with the indicated proteins in EBM alone. Signal intensities were normalized to α-tubulin. A) Levels of ERK1/2 phosphorylation induced by 3 µM S100A4 for the indicated periods of time. B) Levels of ERK1/2 phosphorylation induced by S100A4 (3 µM), VEGF (10 ng/mL) and S100A4 plus VEGF for 15 min. Levels of ERK1/2-dependent S100A4 and VEGF phosphorylation by using the anti-RAGE mAb. C) Levels of ERK1/2 phosphorylation induced by S100A4 (3 µM) for 15 min either in the absence or in the presence of P3. D) Dose-dependent effect of anti-RAGE (10–200 nM) on S100A4-induced KDR expression. E) NF-κB binding in control cells (c) and cells treated (st) with S100A4 (3 µM) and competitions with 2.5X and 5X-fold excess of unlabelled NF-κB ds oligonucleotide. F) Supershift analysis: nuclear extracts were incubated overnight at 4°C in the presence of anti-p50 (arrow) and anti-p65 (arrowhead) antibodies before the addition of the consensus NF-κB probe. G) Gel-shift using the consensus NF-κB probe and nuclear extracts from control HUVEC (c), cells treated (st) with S100A4 (3 µM) or with the anti-RAGE antibody (200 nM), 2 h before S100A4 stimulation.

Journal: PLoS ONE

Article Title: Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

doi: 10.1371/journal.pone.0072480

Figure Lengend Snippet: Cells were treated with the indicated proteins in EBM alone. Signal intensities were normalized to α-tubulin. A) Levels of ERK1/2 phosphorylation induced by 3 µM S100A4 for the indicated periods of time. B) Levels of ERK1/2 phosphorylation induced by S100A4 (3 µM), VEGF (10 ng/mL) and S100A4 plus VEGF for 15 min. Levels of ERK1/2-dependent S100A4 and VEGF phosphorylation by using the anti-RAGE mAb. C) Levels of ERK1/2 phosphorylation induced by S100A4 (3 µM) for 15 min either in the absence or in the presence of P3. D) Dose-dependent effect of anti-RAGE (10–200 nM) on S100A4-induced KDR expression. E) NF-κB binding in control cells (c) and cells treated (st) with S100A4 (3 µM) and competitions with 2.5X and 5X-fold excess of unlabelled NF-κB ds oligonucleotide. F) Supershift analysis: nuclear extracts were incubated overnight at 4°C in the presence of anti-p50 (arrow) and anti-p65 (arrowhead) antibodies before the addition of the consensus NF-κB probe. G) Gel-shift using the consensus NF-κB probe and nuclear extracts from control HUVEC (c), cells treated (st) with S100A4 (3 µM) or with the anti-RAGE antibody (200 nM), 2 h before S100A4 stimulation.

Article Snippet: Dishes were washed eight times with washing buffer (PBS-0.1% Tween-20) and Rabbit polyclonal anti-S100A4 secondary antibody (Dako) at 4 µg/mL was added to the wells which were incubated for 1 h at 37°C.

Techniques: Expressing, Binding Assay, Incubation, Electrophoretic Mobility Shift Assay

A) Molecular interaction between S100A4 and RAGE by SPR. Overlayed sensorgrams of interaction between immobilized RAGE and S100A4 at four different concentrations. Numbers 1 and 2 indicate the start and the end of S100A4 injection, respectively. B) Dose-response inhibition of S100A4-RAGE interaction by 5C3 mAb. Antibody was used at 60–500 nM for 2 µM S100A4.

Journal: PLoS ONE

Article Title: Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

doi: 10.1371/journal.pone.0072480

Figure Lengend Snippet: A) Molecular interaction between S100A4 and RAGE by SPR. Overlayed sensorgrams of interaction between immobilized RAGE and S100A4 at four different concentrations. Numbers 1 and 2 indicate the start and the end of S100A4 injection, respectively. B) Dose-response inhibition of S100A4-RAGE interaction by 5C3 mAb. Antibody was used at 60–500 nM for 2 µM S100A4.

Article Snippet: Dishes were washed eight times with washing buffer (PBS-0.1% Tween-20) and Rabbit polyclonal anti-S100A4 secondary antibody (Dako) at 4 µg/mL was added to the wells which were incubated for 1 h at 37°C.

Techniques: Injection, Inhibition

A) Comparison of tumor growth from M21 cells transfected either with human S100A4 or with mock vector. Groups had 15 animals. Bars of tumor weight show the mean ± SEM. *p<0.05. B) Antitumor activity of 5C3 in M21-S100A4 tumors. PBS (negative control) or 5C3 (25 mg/kg) was given i.p. three times a week (1010100) to 5 animals per group. At the end of the experiment, mice were sacrificed and tumors were weighted. Graphs of RTV show the activity of 5C3 compared with the control group. Bars of tumor weight show the mean ± SEM. *p<0.05 by. C) Immunohistology of tumor microvasculature analyzing CD31 staining. Box and whiskers graphs show the vascular density in a defined tumor area (MVD) expressed as the mean of vascular profiles (v.p.) per mm 2 , and the quantification of vessel area in the tumor (Aa). Quantifications were made from more than 10 pictures per slice at a magnification of X120 (2.4 mm 2 ). Images were analyzed using the NIH ImageJ software. Graphs show the mean ± SEM. ns p>0.05, **p<0.01.

Journal: PLoS ONE

Article Title: Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

doi: 10.1371/journal.pone.0072480

Figure Lengend Snippet: A) Comparison of tumor growth from M21 cells transfected either with human S100A4 or with mock vector. Groups had 15 animals. Bars of tumor weight show the mean ± SEM. *p<0.05. B) Antitumor activity of 5C3 in M21-S100A4 tumors. PBS (negative control) or 5C3 (25 mg/kg) was given i.p. three times a week (1010100) to 5 animals per group. At the end of the experiment, mice were sacrificed and tumors were weighted. Graphs of RTV show the activity of 5C3 compared with the control group. Bars of tumor weight show the mean ± SEM. *p<0.05 by. C) Immunohistology of tumor microvasculature analyzing CD31 staining. Box and whiskers graphs show the vascular density in a defined tumor area (MVD) expressed as the mean of vascular profiles (v.p.) per mm 2 , and the quantification of vessel area in the tumor (Aa). Quantifications were made from more than 10 pictures per slice at a magnification of X120 (2.4 mm 2 ). Images were analyzed using the NIH ImageJ software. Graphs show the mean ± SEM. ns p>0.05, **p<0.01.

Article Snippet: Dishes were washed eight times with washing buffer (PBS-0.1% Tween-20) and Rabbit polyclonal anti-S100A4 secondary antibody (Dako) at 4 µg/mL was added to the wells which were incubated for 1 h at 37°C.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Negative Control, Staining, Software

A) Tumor growth of MiaPACA-2 transfected either with the pSilencer vector encoding for a shRNA against human S100A4 or for a non-related shRNA were compared. Groups had 15 animals. Graphs represent only animals that developed tumors. Bars of tumor weight show the mean ± SEM. ***p<0.001 . B) S100A4 plasma levels in MiaPACA-2 transfected with shRNA-S100A4 or with a non-related shRNA were measured once a week. C) Correlation between plasma levels of S100A4 protein and tumor burden. Graphs of plasma levels show the mean ± SEM. **p<0.01 . r 2 represents the coefficient of determination.

Journal: PLoS ONE

Article Title: Therapeutic Targeting of Tumor Growth and Angiogenesis with a Novel Anti-S100A4 Monoclonal Antibody

doi: 10.1371/journal.pone.0072480

Figure Lengend Snippet: A) Tumor growth of MiaPACA-2 transfected either with the pSilencer vector encoding for a shRNA against human S100A4 or for a non-related shRNA were compared. Groups had 15 animals. Graphs represent only animals that developed tumors. Bars of tumor weight show the mean ± SEM. ***p<0.001 . B) S100A4 plasma levels in MiaPACA-2 transfected with shRNA-S100A4 or with a non-related shRNA were measured once a week. C) Correlation between plasma levels of S100A4 protein and tumor burden. Graphs of plasma levels show the mean ± SEM. **p<0.01 . r 2 represents the coefficient of determination.

Article Snippet: Dishes were washed eight times with washing buffer (PBS-0.1% Tween-20) and Rabbit polyclonal anti-S100A4 secondary antibody (Dako) at 4 µg/mL was added to the wells which were incubated for 1 h at 37°C.

Techniques: Transfection, Plasmid Preparation, shRNA