Journal: International Journal of Molecular Sciences

Article Title: Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells

doi: 10.3390/ijms242316851

Figure Lengend Snippet: Conditioned media from G17-stimulated melanoma cells exhibit upregulation of MMP-2 and downregulation of TIMP-3 expression. Representative demonstration of the confirmation of proteomic data using Western blot analysis, with prosaposin serving as a benchmark for gastrin responsiveness ( A ). Mean and standard deviation (±SD) of the densitometric analysis results from three independent experiments ( B ). Statistical difference (* p < 0.05) between treated and untreated control groups (considered as 100%) are indicated as follows: prosaposin, TIMP-3 and TIMP-1 levels in G361 cells and MMP2 expressions in the SK-MEL-2 cell line.

Article Snippet: Antibodies against MMP-2, TIMP-3 (R&D Systems Abingdon, UK; AF902 and MAB973, respectively), TIMP-1, TIMP-2, prosaposin, and MMP-1 (R&D Systems, Abingdon, UK; AF980, AF971, AF8520, and MAB901, respectively) were utilized.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: The Journal of Biological Chemistry

Article Title: Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice *

doi: 10.1074/jbc.M115.701433

Figure Lengend Snippet: TIMP1 is similarly induced at the protein level in FAPLacZ/LacZversus wild-type mice after intratracheal bleomycin. A, representative Western blot of whole lung homogenates from mice 10 days after intratracheal bleomycin (Bleo) (1.75 IU/kg) versus saline. B, ImageJ densitometry quantification of TIMP1 band intensity in saline versus bleomycin-treated mouse lung homogenates on Western blot, normalized to β-actin. n = 3 mice per saline-treated group; n = 5 mice per bleomycin-treated group.

Article Snippet: Antibodies used were as follows: TIMP1 antibody (R&D Systems; AF980; 0.1 μg/ml); p-Smad2 (Ser-245/-250/-255) (Cell Signaling Technology; catalog no. 3104; 1:500); Smad2/3 (Cell Signaling Technology; catalog no. 5678; 1:500); α-SMA (Abcam; AB5694; 1:5000); type I collagen (EMD Millipore; AB765P; 1:500); sheep anti-human FAP (R&D Systems; AF3715; 0.5 μg/ml); β-actin (Cell Signaling Technology; catalog no. 4967; 1:1000); and GAPDH (Sigma; catalog no. G9545; 1:5000).

Techniques: Western Blot, Saline

Journal: Journal of vascular surgery

Article Title: RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

doi: 10.1016/j.jvs.2017.11.091

Figure Lengend Snippet: Angiotensin II (Ang II)-induced receptor activator of nuclear factor κB ligand (RANKL) expression in smooth muscle cells. Murine vascular smooth muscle cells (MOVAS cells) were stimulated with Ang II. Expression of RANKL messenger RNA (mRNA) and protein was quantified by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Forskolin (FSK) was used as a positive control. Ang II dose dependently induced Rankl mRNA (A) and RANKL protein expression (B). A time-course experiment showed increased Rankl mRNA (C) and RANKL protein (D) from 24 to 72 hours with a maximum at around 48 hours. Relative expression of Western blots was quantified using ImageJ. E and F, Increased RANKL expression through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway in smooth muscle cells. MOVAS cells were stimulated with 1 μM Ang II. JAK2 and STAT5 activities were assayed in Western blots. E, JAK2 and STAT5 phosphorylation was increased at 12 hours. F, Effect of TG101348, a selective inhibitor of JAK2, on Ang II-induced RANKL expression. TG101348 suppressed Ang II-induced RANKL expression in MOVAS cells. G-I, Analysis of the effects of Ang II and RANKL-neutralizing antibody on MOVAS cells. Cells were cultured in growth medium alone or media containing Ang II (1 μM), RANKL-neutralizing antibody (100 ng/mL), or both for 48 hours. G, Mitochondrial membrane potential of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. H, Proliferation of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. OD, optical density. I, Flow cytometric analysis of MOVAS cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. Cells were analyzed for the expression of tissue inhibitor of metalloproteinase 1 (TIMP1), transforming growth factor β (TGF-β), bone morphogenetic protein 4 (BMP4), matrix metalloproteinase (MMP) 9, and MMP2. Flow cytometric data are represented as the mean fluorescence intensity ± standard deviation of four replicates.

Article Snippet: Cells were resuspended in permeabilization buffer containing the following primary antibodies: anti-transforming growth factor β (TGF-β)-Alexa Fluor 700 (IC1835N; R&D Systems, Minneapolis, Minn), anti-bone morphogenetic protein 4 (BMP4)-Alexa Fluor 647 (sc-12721; Santa Cruz Biotechnology), anti-MMP-2-phycoerythrin (sc-13594; Santa Cruz Biotechnology), anti-tissue inhibitor of metalloproteinase 1 (TIMP1)-biotin (BAF980; R&D Systems), anti-RANKL-Alexa Fluor 405 (NB100–56593AF405; Novus Biologicals, Littleton, Colo), and anti-Mmp9- peridinin-chlorophyll (SAB5200309; Sigma-Aldrich).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Positive Control, Phospho-proteomics, Cell Culture, Membrane, Fluorescence, Standard Deviation

Journal: Journal of vascular surgery

Article Title: RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

doi: 10.1016/j.jvs.2017.11.091

Figure Lengend Snippet: Receptor activator of nuclear factor κB ligand (RANKL)-mediated stimulation of macrophages. RAW 264.7 macrophages were cultured with RANKL for 2 days, and matrix metalloproteinase 9 (MMP9) messenger RNA (mRNA) and protein expression was determined in the cell lysates by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. A, Mmp9 mRNA expression was dose dependently increased by RANKL stimulation. B, Representative Western blot and densitometric analysis of MMP9 protein expression in cell lysates of RAW264.7 cells treated with RANKL. Relative expression of Western blots was quantified using ImageJ. C-E, Analysis of the effects of angiotensin II (Ang II) and RANKL-neutralizing antibody on RAW 264.7 cells. Cells were cultured in growth medium alone or media containing Ang II (1 μM), RANKL-neutralizing antibody (100 ng/mL), or both for 48 hours. C, Mitochondrial membrane potential of RAW 264.7 cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. D, Proliferation of RAW 264.7 cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. OD, optical density. E, Flow cytometric analysis of RAW 264.7 cells cultured with Ang II, RANKL-neutralizing antibody, or both for 48 hours. Cells were analyzed for the expression of tissue inhibitor of metalloproteinase 1 (TIMP1), transforming growth factor β (TGF-β), bone morphogenetic protein 4 (BMP4), MMP9, MMP2, and RANKL. Flow cytometric data are represented as the mean fluorescence intensity ± standard deviation of four replicates.

Article Snippet: Cells were resuspended in permeabilization buffer containing the following primary antibodies: anti-transforming growth factor β (TGF-β)-Alexa Fluor 700 (IC1835N; R&D Systems, Minneapolis, Minn), anti-bone morphogenetic protein 4 (BMP4)-Alexa Fluor 647 (sc-12721; Santa Cruz Biotechnology), anti-MMP-2-phycoerythrin (sc-13594; Santa Cruz Biotechnology), anti-tissue inhibitor of metalloproteinase 1 (TIMP1)-biotin (BAF980; R&D Systems), anti-RANKL-Alexa Fluor 405 (NB100–56593AF405; Novus Biologicals, Littleton, Colo), and anti-Mmp9- peridinin-chlorophyll (SAB5200309; Sigma-Aldrich).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Membrane, Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: A hybrid sub-lineage of Listeria monocytogenes comprising hypervirulent isolates

doi: 10.1038/s41467-019-12072-1

Figure Lengend Snippet: WTA galactosylation promotes association of Ami, ActA and InlA to the surface of Lm. a Surface-associated and secreted Lm proteins extracts were obtained from XYSN, ∆1095, and ∆1095::1095. LLO protein levels were used as sample loading control. The image presented is representative of three independent experiments. b Confocal images of XYSN, Δ1095, Δ1095::1095 in Caco-2 BBe cells at 7 h-post infection. ActA was visualized with an anti-ActA monoclonal antibody and goat Anti-Mouse Alexa Fluor 488-conjugated IgG (green), the actin cytoskeleton was stained by phalloidin (red), and DNA was stained by DAPI (blue) following fixation and permeabilization of the sample. Magnification of all images: ×1000. Scale bars, 10 μm

Article Snippet: Proteins were detected using the following antibodies: mouse monoclonal antibody against LLO (3B6) and ActA (6F5) at a 1:2000 dilution, and rabbit polyclonal anti-InlA serum (CSB-PA758331HA01AAD, CUSABIO) at 1:2000 dilution and anti-Ami serum at a 1:5000 dilution – .

Techniques: Control, Infection, Staining

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Schematic representation of LRP-1-derived minireceptors carrying no-ligand-binding cluster (SPCT), extracellular binding-domain II (DII) or extracellular binding-domain IV (DIV). Each construct contains a HA tag at the amino-terminus of the α-chain. B. Transfected CHO cells stably express HA-tagged SPCT (SPCT), HA-tagged mini LRP-II (DII), or HA-tagged mini LRP-IV (DIV). Nontransfected cells served as control (CTRL). Biotinylation of cell-surface proteins was performed, followed by an immunoblot (IB) analysis using anti-HA tag. Bands correspond to the expected molecular weights of SPCT (106 kDa; arrowhead), DII (153 kDa; star), and DIV (164 kDa; double star). C. CHO cells overexpressing HA-tagged LRP-1-derived minireceptors (SPCT, DII, DIV) or not (CTRL) were transiently transfected with RFP-tagged TIMP-1 for 24 hours. Cell-surface proteins were subjected to immunoprecipitation (IP) assay with either anti-HA tag (left panel) or an anti-RFP tag (right panel). Then, immunoblot (IB) analysis was conducted using both anti-LRP-1 β-chain (5A6) and anti-RFP tag. D. Representative sensorgrams for TIMP-1 interacting with DII (left panel) and DIV (right panel). A set of concentrations (5–80 nM) of TIMP-1 or EGF was sequentially injected over immobilized Fc-DII and Fc-DIV. The solid black lines represent the specific binding of TIMP-1 obtained after double-subtraction of the signal obtained on the control flow cell and a blank run. The dotted black lines represent the fit of the data with a kinetic titration 1∶1 interaction model. The grey lines represent the specific binding of EGF obtained after double-subtraction of the signal obtained on the control flow cell and a blank run. Arrows indicate the beginning of each injection. The data illustrated are representative of three independent experiments. E. Binding and internalization of exogenous fluorescent TIMP-1 (fluo-TIMP-1) by CHO cells overexpressing minireceptor SPCT (left), DII (middle) or DIV (right). Binding was assessed by incubating fluo-TIMP-1 (10 nM) at 4°C for 2 hours. Cells were then transferred to 37°C for additional 2 h to allow internalization. All incubations were performed with or without RAP (500 nM), an antagonist of LRP-1-mediated binding and consequently, endocytosis. Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 10 A.U. are considered to be nonspecific. Images in B–D are representative of results obtained in 3 independent experiments. Values in E represent the means ± s.e.m. of 3 independent experiments. NS, not significant; * p <0.05, as compared to untreated cells.

Article Snippet: Mouse recombinant TIMP-1 and goat anti-mouse TIMP-1 antibodies were purchased from R&D System (Minneapolis, MN, USA).

Techniques: Derivative Assay, Ligand Binding Assay, Binding Assay, Construct, Transfection, Stable Transfection, Western Blot, Immunoprecipitation, Injection, Titration, Fluorescence, Spectrophotometry

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were plated onto poly-L-lysine-coated coverslips for 24 h at 37°C, fixed, washed and stained with anti-LRP1 antibody (Alexa Fluor 488, green) and anti-TIMP-1 antibody (Alexa Fluor 568, red) before confocal microscopy analysis. Nuclei were counterstained with DAPI (blue) and appropriate secondary antibody controls were performed. LRP-1 labeling (left), TIMP-1 labeling (middle), and a merged image (right) are shown. B. Biotinylation of cell-surface proteins was conducted at 4°C from cortical neurons previously treated for 24 h with or without RAP (500 nM). Proteins were affinity precipitated with avidin-agarose beads, then LRP-1-containing complexes were immunoprecipitated by either anti-LRP-1 β-chain (LRP-1 β; left panel) or anti-LRP-1 α-chain (LRP-1 α; middle panel) and analyzed by western-blot using anti-LRP-1 β-chain (5A6), anti-LRP-1 α-chain (8G1) and anti-TIMP-1 antibodies. Nonspecific IgGs were used as a negative control of immunoprecipitation. The presence of TIMP-1 in immunocomplexes was quantified by densitometric analysis relative to immunoprecipitated LRP-1-α-chain (histogram, right panel). C. Binding and internalization of exogenous fluorescent TIMP-1 (fluo-TIMP-1) by cortical neurons. Binding was determined by incubating fluo-TIMP-1 at 4°C for 2 h. After extensive washes, part of the cells was used to quantify total binding. The other part was incubated at 37°C for an additional 1 h to permit endocytosis. Experiments were carried out with or without RAP (500 nM). Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 10 A.U. are considered to be nonspecific. Images in A and B are representative of results obtained in 3 independent experiments. Values in B and C represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01, as compared to untreated cells. Scale bar: 5 µm.

Article Snippet: Mouse recombinant TIMP-1 and goat anti-mouse TIMP-1 antibodies were purchased from R&D System (Minneapolis, MN, USA).

Techniques: Staining, Confocal Microscopy, Labeling, Avidin-Biotin Assay, Immunoprecipitation, Western Blot, Negative Control, Binding Assay, Incubation, Fluorescence, Spectrophotometry

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were cultured for 24-L-lysine-coated coverslips and then treated for 30 min with TIMP-1 (10 nM), RAP (500 nM), blocking LRP-1 polyclonal antibodies (R2629) or a combination of TIMP-1+RAP and TIMP-1+R2629. Untreated cells served as control (CTRL). Cells were labeled with anti-βIII-tubulin monoclonal antibody and observed under confocal microscopy. B. Quantification of neurite mean length per cell was performed using the ImageJ plugin NeuronJ and expressed as percent of untreated neurons (CTRL). Images in A are representative of results obtained in 3 independent experiments. Values in B represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01. Scale bar: 10 µm.

Article Snippet: Mouse recombinant TIMP-1 and goat anti-mouse TIMP-1 antibodies were purchased from R&D System (Minneapolis, MN, USA).

Techniques: Cell Culture, Blocking Assay, Labeling, Confocal Microscopy

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were treated after 24-L-lysine-coated coverslips for 30 min with TIMP-1 (10 nM), RAP (500 nM) or blocking LRP-1 polyclonal antibodies (R2629) or a combination of TIMP-1+RAP and TIMP-1+R2629. Untreated cells served as a control (CTRL). Neurons were incubated with Alexa Fluor 568-phalloidin to label F-actin structures and analyzed by confocal microscopy. B. 3D-quantification of growth cone volume was performed using the AMIRA software and expressed as percent of untreated neurons (CTRL). Images in A are representative of results obtained in 3 independent experiments. Values in B represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01. Scale bar: 5 µm.

Article Snippet: Mouse recombinant TIMP-1 and goat anti-mouse TIMP-1 antibodies were purchased from R&D System (Minneapolis, MN, USA).

Techniques: Blocking Assay, Incubation, Confocal Microscopy, Software

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were allowed to grow during 24-L-lysine-coated coverslips, and treated for 30 min with FLAG-TIMP-1 (10 nM) or FLAG-T2G (10 nM). Neurons were then stained with anti-LRP-1 antibody (Alexa Fluor 568, red) or anti-FLAG antibody (Alexa Fluor 488, green) and analyzed by confocal microscopy. Nuclei were counterstained with DAPI (blue). Images were treated with the AMIRA sofware. Fluorescent signals corresponding to LRP-1, FLAG and colocalization were shown by red (left), green (middle) and cyan (right) labeling. B–C. Neurons were treated as indicated in A , in the absence or presence of RAP. B. Neurites were labeled with anti-βIII-tubulin antibody and observed under confocal microscopy. The neurite mean length per cell was determined using the ImageJ plugin NeuronJ and expressed as percent of untreated neurons (CTRL). C. Actin-rich growth cones were visualized with Alexa Fluor 568-phalloidin, observed under confocal microscopy and quantified using the AMIRA software (right panel). Images in A are representative of results obtained in 3 independent experiments. Values in B and C represent the mean ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01. Scale bar: 5 µm.

Article Snippet: Mouse recombinant TIMP-1 and goat anti-mouse TIMP-1 antibodies were purchased from R&D System (Minneapolis, MN, USA).

Techniques: Staining, Confocal Microscopy, Labeling, Software