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R&D Systems human timp 1 quantikine elisa kit
Endogenous <t>TIMP-1</t> interacts with LRP-1 in cortical neurons. 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
Human Timp 1 Quantikine Elisa Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0103839

Endogenous TIMP-1 interacts with LRP-1 in cortical neurons. 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
Figure Legend Snippet: Endogenous TIMP-1 interacts with LRP-1 in cortical neurons. 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

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

TIMP-1 binding to LRP-1 reduces neurite length. 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
Figure Legend Snippet: TIMP-1 binding to LRP-1 reduces neurite length. 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

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

TIMP-1 binding to LRP-1 increases growth cone volume. 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
Figure Legend Snippet: TIMP-1 binding to LRP-1 increases growth cone volume. 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

Techniques Used: Binding Assay, Blocking Assay, Incubation, Confocal Microscopy, Software

Inactive T2G mutant of TIMP-1 colocalizes with LRP-1 and exerts similar effects on the morphology of cortical neurons than wild-type TIMP-1. 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
Figure Legend Snippet: Inactive T2G mutant of TIMP-1 colocalizes with LRP-1 and exerts similar effects on the morphology of cortical neurons than wild-type TIMP-1. 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

Techniques Used: Mutagenesis, Staining, Confocal Microscopy, Labeling, Software

Domains II and IV of the extracellular α-chain of LRP-1 are required to bind and promote TIMP-1 endocytosis in CHO cells. 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
Figure Legend Snippet: Domains II and IV of the extracellular α-chain of LRP-1 are required to bind and promote TIMP-1 endocytosis in CHO cells. 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

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

2) Product Images from "Modelling human liver fibrosis in the context of non-alcoholic steatohepatitis using a microphysiological system"

Article Title: Modelling human liver fibrosis in the context of non-alcoholic steatohepatitis using a microphysiological system

Journal: Communications Biology

doi: 10.1038/s42003-021-02616-x

Liver MPS NASH model demonstrates fibrotic phenotype. PHH alone (Control and Steatosis) or PHH, KC and HSC co-cultures (NASH) were cultured in the MPS platform for 14 days under standard media (control) or high fat conditions (Steatosis and NASH). a Liver microtissues were stained for cytoskeleton (phalloidin), collagen-type I and α-SMA and imaged by confocal microscopy. Representative images shown and scale bars 200 µm. b Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all microtissues within a FOV (min 8, max 12) and two FOV per scaffold. c Secreted fibrosis biomarkers TIMP-1, Pro-collagen 1, YKL-40 and Fibronectin were all measured in cell culture medium by ELISA, at the end of the culture. d The expression of fibrosis genes was analysed in total RNA from steatosis and NASH models using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression levels are expressed as Log10 relative expression compared to housekeeping genes (GAPDH/B2M/HPRT1) and compared between the NASH and steatosis models (red = upregulation, green = downregulation, black = no change, upper line—1.5-fold increase, lower line 1.5-fold decrease). All data points shown with means ± SD highlighted, data generated from a minimum of nine independent cultures (three donors per condition and n = 3 per donor).
Figure Legend Snippet: Liver MPS NASH model demonstrates fibrotic phenotype. PHH alone (Control and Steatosis) or PHH, KC and HSC co-cultures (NASH) were cultured in the MPS platform for 14 days under standard media (control) or high fat conditions (Steatosis and NASH). a Liver microtissues were stained for cytoskeleton (phalloidin), collagen-type I and α-SMA and imaged by confocal microscopy. Representative images shown and scale bars 200 µm. b Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all microtissues within a FOV (min 8, max 12) and two FOV per scaffold. c Secreted fibrosis biomarkers TIMP-1, Pro-collagen 1, YKL-40 and Fibronectin were all measured in cell culture medium by ELISA, at the end of the culture. d The expression of fibrosis genes was analysed in total RNA from steatosis and NASH models using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression levels are expressed as Log10 relative expression compared to housekeeping genes (GAPDH/B2M/HPRT1) and compared between the NASH and steatosis models (red = upregulation, green = downregulation, black = no change, upper line—1.5-fold increase, lower line 1.5-fold decrease). All data points shown with means ± SD highlighted, data generated from a minimum of nine independent cultures (three donors per condition and n = 3 per donor).

Techniques Used: Cell Culture, Staining, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Polymerase Chain Reaction, Generated

Obeticholic acid and Elafibranor both modulate inflammatory and fibrosis phenotype in liver MPS NASH model and phenotype can additionally be reversed with dietary changes. PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat conditions and dosed with varying concentrations of Obeticholic acid (OCA) and Elafibranor (ELF) QD or vehicle control (control) for 10 days, following an initial 4-day pre-culture phase. a The secreted cytokine profile from liver microtissues was compared by Luminex to determine the effects of each compound on the inflammatory profile of the liver with samples analysed at day 8 and 14 of the culture. Data were normalised by Z-transformation to allow comparison of all analytes and presented as a heatmap. b The expression of fibrosis-associated genes was analysed in total RNA from control, OCA and ELF treated liver microtissues (highest concentration for each compound) using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression changes are expressed as a fold change over control. c Liver microtissues were stained for collagen-type I, α-SMA, nuclei (DAPI - blue), phalloidin (green) and imaged by confocal microscopy. Representative images are shown and scale bars 200 µm. d Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all the microtissues imaged within an MPS scaffold (min 8, max 20 microtissues). e PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat or lean conditions for 30 days. Cultures either remained in the same type of media throughout or were switched at day 15 from fat media to lean media (Fat - > Lean). Cell culture medium samples were analysed for the presence of IL-6, f MCP-1, g TIMP-1, which were measured by ELISA. h Microtissues were stained with Oil-Red O to determine fat loading during culture period, total stain was quantified by absorbance at 510 nm. All datapoints shown, either as box-whisker plots highlighting mean and min-max or with error bars highlighting means ± SD. All data from a minimum of three independent cultures; statistical comparisons made to control samples unless other comparison shown.
Figure Legend Snippet: Obeticholic acid and Elafibranor both modulate inflammatory and fibrosis phenotype in liver MPS NASH model and phenotype can additionally be reversed with dietary changes. PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat conditions and dosed with varying concentrations of Obeticholic acid (OCA) and Elafibranor (ELF) QD or vehicle control (control) for 10 days, following an initial 4-day pre-culture phase. a The secreted cytokine profile from liver microtissues was compared by Luminex to determine the effects of each compound on the inflammatory profile of the liver with samples analysed at day 8 and 14 of the culture. Data were normalised by Z-transformation to allow comparison of all analytes and presented as a heatmap. b The expression of fibrosis-associated genes was analysed in total RNA from control, OCA and ELF treated liver microtissues (highest concentration for each compound) using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression changes are expressed as a fold change over control. c Liver microtissues were stained for collagen-type I, α-SMA, nuclei (DAPI - blue), phalloidin (green) and imaged by confocal microscopy. Representative images are shown and scale bars 200 µm. d Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all the microtissues imaged within an MPS scaffold (min 8, max 20 microtissues). e PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat or lean conditions for 30 days. Cultures either remained in the same type of media throughout or were switched at day 15 from fat media to lean media (Fat - > Lean). Cell culture medium samples were analysed for the presence of IL-6, f MCP-1, g TIMP-1, which were measured by ELISA. h Microtissues were stained with Oil-Red O to determine fat loading during culture period, total stain was quantified by absorbance at 510 nm. All datapoints shown, either as box-whisker plots highlighting mean and min-max or with error bars highlighting means ± SD. All data from a minimum of three independent cultures; statistical comparisons made to control samples unless other comparison shown.

Techniques Used: Cell Culture, Luminex, Transformation Assay, Expressing, Concentration Assay, Polymerase Chain Reaction, Staining, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay, Whisker Assay

3) Product Images from "TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients"

Article Title: TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients

Journal: Frontiers in Medicine

doi: 10.3389/fmed.2021.774623

The results of TIMP-1 (A) and TSP-1 (B) between COPD and COPD-PH group in validation cohort by ELISA. The P -value of each factor was obtained from unpaired Student's t -test analysis. ** P
Figure Legend Snippet: The results of TIMP-1 (A) and TSP-1 (B) between COPD and COPD-PH group in validation cohort by ELISA. The P -value of each factor was obtained from unpaired Student's t -test analysis. ** P

Techniques Used: Enzyme-linked Immunosorbent Assay

Summary figure of the schematic workflow of the current study. (A) Initial screening of angiogenic factors and cytokines. (B) ELISA verification in validation cohort and related statistical analysis. (C) Involvement of TIMP-1 in the hPASMCs mechanism. COPD, Chronic obstructive pulmonary disease; COPD-PH, Chronic obstructive pulmonary disease associated pulmonary hypertension; CSE, Cigarette smoke exposure; ELISA, Enzyme-Linked Immunosorbent Assay; hPASMCs, Human Pulmonary Artery Smooth Muscle Cells; TIMP-1, Tissue Inhibitor of Metalloproteinases-1. This figure was created using BioRender .
Figure Legend Snippet: Summary figure of the schematic workflow of the current study. (A) Initial screening of angiogenic factors and cytokines. (B) ELISA verification in validation cohort and related statistical analysis. (C) Involvement of TIMP-1 in the hPASMCs mechanism. COPD, Chronic obstructive pulmonary disease; COPD-PH, Chronic obstructive pulmonary disease associated pulmonary hypertension; CSE, Cigarette smoke exposure; ELISA, Enzyme-Linked Immunosorbent Assay; hPASMCs, Human Pulmonary Artery Smooth Muscle Cells; TIMP-1, Tissue Inhibitor of Metalloproteinases-1. This figure was created using BioRender .

Techniques Used: Enzyme-linked Immunosorbent Assay

Angiogenesis array data. (A) Representative image of angiogenesis array of the plasma from COPD patients without PH [COPD, (a)] and with PH [COPD-PH, (b)] array blots. The levels of angiogenic factors are determined based on their blotting intensity in duplicates. (B–E) Relative protein level of for angiogenesis-associated protein determined by angiogenesis assay (Data were normalized to three pairs of reference spots). (F,G) Based on two-sample independent Student's t -test analysis, TIMP-1 and TSP-1 were significantly higher in COPD-PH than COPD group. Data were present as mean ± SEM ( n = 4). * P
Figure Legend Snippet: Angiogenesis array data. (A) Representative image of angiogenesis array of the plasma from COPD patients without PH [COPD, (a)] and with PH [COPD-PH, (b)] array blots. The levels of angiogenic factors are determined based on their blotting intensity in duplicates. (B–E) Relative protein level of for angiogenesis-associated protein determined by angiogenesis assay (Data were normalized to three pairs of reference spots). (F,G) Based on two-sample independent Student's t -test analysis, TIMP-1 and TSP-1 were significantly higher in COPD-PH than COPD group. Data were present as mean ± SEM ( n = 4). * P

Techniques Used: Angiogenesis Assay

CSE increased TIMP-1 level in cultured hPASMCs, and TIMP-1 decreased hPASMCs proliferation under CSE. (A) Western Blotting indicates 0.5% CSE treatment for 36 h increased TIMP-1 expression in hPASMCs. (B) TIMP-1 attenuated CSE-stimulated hPASMCs proliferation. CSE, Cigarette Smoking Extract; hPASMCs, human Pulmonary Arterial Smooth Muscle Cell, ** P
Figure Legend Snippet: CSE increased TIMP-1 level in cultured hPASMCs, and TIMP-1 decreased hPASMCs proliferation under CSE. (A) Western Blotting indicates 0.5% CSE treatment for 36 h increased TIMP-1 expression in hPASMCs. (B) TIMP-1 attenuated CSE-stimulated hPASMCs proliferation. CSE, Cigarette Smoking Extract; hPASMCs, human Pulmonary Arterial Smooth Muscle Cell, ** P

Techniques Used: Cell Culture, Western Blot, Expressing

4) Product Images from "IL-11 is a crucial determinant of cardiovascular fibrosis"

Article Title: IL-11 is a crucial determinant of cardiovascular fibrosis

Journal: Nature

doi: 10.1038/nature24676

IL-11 activates fibroblasts and is required for the pro-fibrotic effect of TGFβ1 a , b , High-resolution fluorescence imaging after TGFβ1 or IL-11 treatment (5 ng ml −1 , 24 h) of primary cardiac fibroblasts. Immunostaining of nuclei (DAPI, blue), ACTA2 (red) and F-actin (phalloidin, green) indicated that both TGFβ1 and IL-11 activate fibroblast stress fibre formation and increase the number of myofibroblasts in vitro to similar levels. Experiment was repeated four times with similar results. c , Automated quantification of fluorescence (Operetta assay n = 7 measurements per n = 6 independent experiments) of primary atrial fibroblasts reveals significant fibroblast activation and ECM production induced by both TGFβ1 and IL-11 (5 ng ml −1 , 24 h). d , In addition, TGFβ1 effects can be reduced with an anti-IL-11 antibody (2 μg ml −1 ). c , d , Collagen secretion in the supernatant ( n = 6 independent experiments) was assessed with Sirius Red. e , Mouse primary fibroblasts were incubated for 24 h with indicated concentrations of recombinant human or mouse IL-11. Fibroblast activation was monitored using the Operetta High-Content Imaging platform and immunostaining for ACTA2. rhIL-11 was found to inefficiently activate mouse fibroblasts (rmIL-11, n = 2, rhIL-11, n = 4 biologically independent samples) compared to rmIL-11; this occurred for rhIL-11 treatment with rhIL-11 from two separate suppliers. f , g , MMP-2 ( f ) and TIMP-1 ( g ) concentration in the supernatant (ELISA) of cardiac fibroblasts ( n = 4 biologically independent samples) without stimulus (−), with TGFβ1 or IL-11 (5 ng ml −1 , 24 h). h , Il-11-neutralizing antibodies (anti-IL-11, 2 μg ml −1 ) block the increase in MMP-2 and TIMP-1 protein. i , In vitro monolayer scratch wound assay of cardiac fibroblasts. Wound closure was compared between stimulated (TGFβ1 or IL-11; 5 ng ml −1 , 24 h) and unstimulated cardiac fibroblasts ( n = 5 biologically independent samples) after 24 h. j , Cardiac fibroblasts ( n = 3 biologically independent samples) were seeded in collagen gel and the contraction was monitored. The area of contraction is compared between stimulated (TGFβ1 or IL-11; 5 ng ml −1 ) and unstimulated groups after 72 h. k , Trans-well migration assay. After 24 h of stimulation (TGFβ1 or IL-11; 5 ng ml −1 ), cardiac fibroblasts ( n = 6 biologically independent samples) that crossed the membrane towards either a TGFβ1- or IL-11-containing compartment were colourimetrically quantified and compared to data from unstimulated cells. l – n , Cardiac fibroblasts were incubated with TGFβ1 (5 ng ml −1 , 24 h) and indicated amounts of IL11RA:gp130 decoy receptors ( l ; 33 amino acid (aa) or 50 aa linker peptide), anti-IL11RA antibody ( m ; 2 μg ml −1 ) or siRNA pools against IL-11 or IL11RA ( n ). l – n , Fibroblast activation was monitored via immunostaining for ACTA2 on the Operetta platform. decoy receptors ( l ): n = 7 measurements per n = 2 independent experiments; anti- IL11RA ( m ): n = 7 measurements per n = 2 independent experiments; siRNA ( n ): Operetta assay n = 7 measurements per n = 10 independent experiments. o , Human renal fibroblasts were incubated with TGFβ1 or IL-11 (5 ng ml −1 , 24 h) in the presence or absence of anti-IL-11 or an IgG control antibodies (2 μg ml −1 each) for 24 h. ECM was assessed using the Operetta platform by staining for collagen I. Fluorescence was normalized to non-stimulated cells (black). p , These results were confirmed with Sirius red assay of the total collagen in the supernatant. q , rmIl-11 stimulation (5 ng ml −1 , 24 h) also activated mouse cardiac and renal fibroblasts. Myofibroblasts and ECM were assessed using the Operetta platform by staining for ACTA2, collagen I or POSTN. Fluorescence was normalized to non-stimulated cells (black). o – q , These experiments were repeated three times with similar results. r , Cardiac fibroblasts analysed on the Operetta high-content imaging platform with immunostaining of ACTA2 after 24 h incubation without stimulus, TGFβ1 (5 ng ml −1 , 24h) or TGFβ1 and IL-6-neutralizing antibody (2 μg ml −1 , 24h). Automated quantification of fluorescence (Operetta assay n = 7 measurements per n = 6 independent experiments) shows no significant decrease in fibroblast activation using anti-IL-6 antibodies. Data are mean and circles show individual values ( e ) or mean ± s.d. and circles show individual values ( c, d bottom right, f – h , k ); box-and-whisker plots ( c , d , l – n , r ) show median (middle line), 25th–75th percentiles (box) and 10th–90th percentiles (whiskers). Two-tailed Dunnett’s test ( c , f , g , i – k ), two-tailed Student’s t -test ( d , h , r ) or two-tailed, Sidak-corrected Student’s t -test ( l – n ). * P
Figure Legend Snippet: IL-11 activates fibroblasts and is required for the pro-fibrotic effect of TGFβ1 a , b , High-resolution fluorescence imaging after TGFβ1 or IL-11 treatment (5 ng ml −1 , 24 h) of primary cardiac fibroblasts. Immunostaining of nuclei (DAPI, blue), ACTA2 (red) and F-actin (phalloidin, green) indicated that both TGFβ1 and IL-11 activate fibroblast stress fibre formation and increase the number of myofibroblasts in vitro to similar levels. Experiment was repeated four times with similar results. c , Automated quantification of fluorescence (Operetta assay n = 7 measurements per n = 6 independent experiments) of primary atrial fibroblasts reveals significant fibroblast activation and ECM production induced by both TGFβ1 and IL-11 (5 ng ml −1 , 24 h). d , In addition, TGFβ1 effects can be reduced with an anti-IL-11 antibody (2 μg ml −1 ). c , d , Collagen secretion in the supernatant ( n = 6 independent experiments) was assessed with Sirius Red. e , Mouse primary fibroblasts were incubated for 24 h with indicated concentrations of recombinant human or mouse IL-11. Fibroblast activation was monitored using the Operetta High-Content Imaging platform and immunostaining for ACTA2. rhIL-11 was found to inefficiently activate mouse fibroblasts (rmIL-11, n = 2, rhIL-11, n = 4 biologically independent samples) compared to rmIL-11; this occurred for rhIL-11 treatment with rhIL-11 from two separate suppliers. f , g , MMP-2 ( f ) and TIMP-1 ( g ) concentration in the supernatant (ELISA) of cardiac fibroblasts ( n = 4 biologically independent samples) without stimulus (−), with TGFβ1 or IL-11 (5 ng ml −1 , 24 h). h , Il-11-neutralizing antibodies (anti-IL-11, 2 μg ml −1 ) block the increase in MMP-2 and TIMP-1 protein. i , In vitro monolayer scratch wound assay of cardiac fibroblasts. Wound closure was compared between stimulated (TGFβ1 or IL-11; 5 ng ml −1 , 24 h) and unstimulated cardiac fibroblasts ( n = 5 biologically independent samples) after 24 h. j , Cardiac fibroblasts ( n = 3 biologically independent samples) were seeded in collagen gel and the contraction was monitored. The area of contraction is compared between stimulated (TGFβ1 or IL-11; 5 ng ml −1 ) and unstimulated groups after 72 h. k , Trans-well migration assay. After 24 h of stimulation (TGFβ1 or IL-11; 5 ng ml −1 ), cardiac fibroblasts ( n = 6 biologically independent samples) that crossed the membrane towards either a TGFβ1- or IL-11-containing compartment were colourimetrically quantified and compared to data from unstimulated cells. l – n , Cardiac fibroblasts were incubated with TGFβ1 (5 ng ml −1 , 24 h) and indicated amounts of IL11RA:gp130 decoy receptors ( l ; 33 amino acid (aa) or 50 aa linker peptide), anti-IL11RA antibody ( m ; 2 μg ml −1 ) or siRNA pools against IL-11 or IL11RA ( n ). l – n , Fibroblast activation was monitored via immunostaining for ACTA2 on the Operetta platform. decoy receptors ( l ): n = 7 measurements per n = 2 independent experiments; anti- IL11RA ( m ): n = 7 measurements per n = 2 independent experiments; siRNA ( n ): Operetta assay n = 7 measurements per n = 10 independent experiments. o , Human renal fibroblasts were incubated with TGFβ1 or IL-11 (5 ng ml −1 , 24 h) in the presence or absence of anti-IL-11 or an IgG control antibodies (2 μg ml −1 each) for 24 h. ECM was assessed using the Operetta platform by staining for collagen I. Fluorescence was normalized to non-stimulated cells (black). p , These results were confirmed with Sirius red assay of the total collagen in the supernatant. q , rmIl-11 stimulation (5 ng ml −1 , 24 h) also activated mouse cardiac and renal fibroblasts. Myofibroblasts and ECM were assessed using the Operetta platform by staining for ACTA2, collagen I or POSTN. Fluorescence was normalized to non-stimulated cells (black). o – q , These experiments were repeated three times with similar results. r , Cardiac fibroblasts analysed on the Operetta high-content imaging platform with immunostaining of ACTA2 after 24 h incubation without stimulus, TGFβ1 (5 ng ml −1 , 24h) or TGFβ1 and IL-6-neutralizing antibody (2 μg ml −1 , 24h). Automated quantification of fluorescence (Operetta assay n = 7 measurements per n = 6 independent experiments) shows no significant decrease in fibroblast activation using anti-IL-6 antibodies. Data are mean and circles show individual values ( e ) or mean ± s.d. and circles show individual values ( c, d bottom right, f – h , k ); box-and-whisker plots ( c , d , l – n , r ) show median (middle line), 25th–75th percentiles (box) and 10th–90th percentiles (whiskers). Two-tailed Dunnett’s test ( c , f , g , i – k ), two-tailed Student’s t -test ( d , h , r ) or two-tailed, Sidak-corrected Student’s t -test ( l – n ). * P

Techniques Used: Fluorescence, Imaging, Immunostaining, In Vitro, Activation Assay, Incubation, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Scratch Wound Assay Assay, Migration, Staining, Whisker Assay, Two Tailed Test

5) Product Images from "Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons"

Article Title: Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons

Journal: Scientific Reports

doi: 10.1038/s41598-017-05039-z

Production of TIMP-1 mutants. ( a ) Schematic representation of T1-WT, T1-F12A and T1-K47A proteins. ( b ) A representative western blot with the indicated antibodies of cell extracts and conditioned medium (cond. med) of stably transfected CHO cells with p3X-FLAG-CMV-14 expressing T1-WT, T1-F12A or T1-K47A. The relative protein level based on three independent experiments is indicated under each band. The gels were run under the same experimental conditions and were shown as cropped gels/blots (Cropped gels/blots are shown in Supplementary Figure S1 ).
Figure Legend Snippet: Production of TIMP-1 mutants. ( a ) Schematic representation of T1-WT, T1-F12A and T1-K47A proteins. ( b ) A representative western blot with the indicated antibodies of cell extracts and conditioned medium (cond. med) of stably transfected CHO cells with p3X-FLAG-CMV-14 expressing T1-WT, T1-F12A or T1-K47A. The relative protein level based on three independent experiments is indicated under each band. The gels were run under the same experimental conditions and were shown as cropped gels/blots (Cropped gels/blots are shown in Supplementary Figure S1 ).

Techniques Used: Western Blot, Stable Transfection, Transfection, Expressing

Identification of residues involved in TIMP-1 interdomain movements. ( a ) Superposition of the deformation energy (dotted line) and atomic fluctuation (coloured bars) data from TIMP-1 residues (numbering of the mature secreted protein) along mode 7. The green arrows point out residues or sets of residues with both high deformation energy and low Cα atomic fluctuation. ( b ) Localisation in the TIMP-1 3D structure of the residues identified in ( a ) with both high deformation energy and low Cα atomic fluctuation. These residues are in an area surrounded by a dotted line. ( c ) Localisation in the TIMP-1 secondary structure of the residues identified in ( a ) with both high deformation energy and low Cα atomic fluctuation (green arrows). α helices are defined by the purple area and β sheets by the yellow area. ( d ) Left: localisation in the TIMP-1 3D structure of F12 and K47 residues. Right: hydrogen bond formed between the carbonyl oxygen of the F12 backbone and the K47 side chain.
Figure Legend Snippet: Identification of residues involved in TIMP-1 interdomain movements. ( a ) Superposition of the deformation energy (dotted line) and atomic fluctuation (coloured bars) data from TIMP-1 residues (numbering of the mature secreted protein) along mode 7. The green arrows point out residues or sets of residues with both high deformation energy and low Cα atomic fluctuation. ( b ) Localisation in the TIMP-1 3D structure of the residues identified in ( a ) with both high deformation energy and low Cα atomic fluctuation. These residues are in an area surrounded by a dotted line. ( c ) Localisation in the TIMP-1 secondary structure of the residues identified in ( a ) with both high deformation energy and low Cα atomic fluctuation (green arrows). α helices are defined by the purple area and β sheets by the yellow area. ( d ) Left: localisation in the TIMP-1 3D structure of F12 and K47 residues. Right: hydrogen bond formed between the carbonyl oxygen of the F12 backbone and the K47 side chain.

Techniques Used:

Confocal analysis of LRP-1-mediated endocytosis. Cortical neurons from mouse embryos cultured for 24 h on poly-L-lysine-coated coverslips were incubated at 4 °C for 30 min with 5 nM T1-WT, T1-F12A or T1K47A in the presence or absence of 500 nM RAP and then incubated at 37 °C for 10 min. Cells were stained with Alexa Fluor 488 for TIMP-1 and Alexa Fluor 568 for EEA1 and analysed by confocal microscopy. Images were treated with AMIRA software. Maximal Intensity Projection (MIP) in greyscale of TIMP-1 labelling (first panel) and EEA1 labelling (second panel), TIMP-1/EEA1 merge (third panel, TIMP-1 in red and EEA1 in green), differential interference contrast (DIC) and isosurface representation of TIMP-1/EEA1 colocalisation (fourth panel) are shown. Images are representative of more than three distinct experiments. Scale bar: 10 µm.
Figure Legend Snippet: Confocal analysis of LRP-1-mediated endocytosis. Cortical neurons from mouse embryos cultured for 24 h on poly-L-lysine-coated coverslips were incubated at 4 °C for 30 min with 5 nM T1-WT, T1-F12A or T1K47A in the presence or absence of 500 nM RAP and then incubated at 37 °C for 10 min. Cells were stained with Alexa Fluor 488 for TIMP-1 and Alexa Fluor 568 for EEA1 and analysed by confocal microscopy. Images were treated with AMIRA software. Maximal Intensity Projection (MIP) in greyscale of TIMP-1 labelling (first panel) and EEA1 labelling (second panel), TIMP-1/EEA1 merge (third panel, TIMP-1 in red and EEA1 in green), differential interference contrast (DIC) and isosurface representation of TIMP-1/EEA1 colocalisation (fourth panel) are shown. Images are representative of more than three distinct experiments. Scale bar: 10 µm.

Techniques Used: Cell Culture, Incubation, Staining, Confocal Microscopy, Software

Quantification of TIMP-1 and EEA1 labelling. ( a ) Quantification of average intensity of TIMP-1 labelling per µm 2 for each cell (n > 10) using ImageJ software. The data are representative of at least three independent experiments with at least five mice embryos. Error bars indicate mean ± SD and **** indicates significantly different from T1-WT without RAP and with a P-value
Figure Legend Snippet: Quantification of TIMP-1 and EEA1 labelling. ( a ) Quantification of average intensity of TIMP-1 labelling per µm 2 for each cell (n > 10) using ImageJ software. The data are representative of at least three independent experiments with at least five mice embryos. Error bars indicate mean ± SD and **** indicates significantly different from T1-WT without RAP and with a P-value

Techniques Used: Software, Mouse Assay

Principal component analysis derived from molecular dynamics simulation of TIMP-1. For each PC, the contribution of each residue to the overall atomic displacement was determined. The green arrows point out residues F12 and K47.
Figure Legend Snippet: Principal component analysis derived from molecular dynamics simulation of TIMP-1. For each PC, the contribution of each residue to the overall atomic displacement was determined. The green arrows point out residues F12 and K47.

Techniques Used: Derivative Assay

Effect of TIMP-1 mutants on neurite outgrowth. ( a ) Immunofluorescence of cortical neurons from mouse embryos cultured for 24 h on poly-L-lysine-coated coverslips and then treated for 30 min with PBS (CTRL) or 5 nM T1-WT, T1-F12A and T1K47A. Cells were labelled with anti-βIII-tubulin monoclonal antibody and observed by fluorescent microscopy. The images are representative of more than three distinct experiments. Scale bar: 10 µm. ( b ) Quantification of neurite mean length per cell performed using the ImageJ plugin NeuronJ. For each group, at least 20 randomly selected fields were analysed in three independent experiments (n = 20). The data are representative of at least three independent experiments with at least five mice embryos. Error bars indicate mean ± SD, *** indicates P-value
Figure Legend Snippet: Effect of TIMP-1 mutants on neurite outgrowth. ( a ) Immunofluorescence of cortical neurons from mouse embryos cultured for 24 h on poly-L-lysine-coated coverslips and then treated for 30 min with PBS (CTRL) or 5 nM T1-WT, T1-F12A and T1K47A. Cells were labelled with anti-βIII-tubulin monoclonal antibody and observed by fluorescent microscopy. The images are representative of more than three distinct experiments. Scale bar: 10 µm. ( b ) Quantification of neurite mean length per cell performed using the ImageJ plugin NeuronJ. For each group, at least 20 randomly selected fields were analysed in three independent experiments (n = 20). The data are representative of at least three independent experiments with at least five mice embryos. Error bars indicate mean ± SD, *** indicates P-value

Techniques Used: Immunofluorescence, Cell Culture, Microscopy, Mouse Assay

Normal mode analysis of TIMP-1 structure. ( a ) Intrinsic dynamic of TIMP-1 along modes 7, 8 and 9. Open structures of TIMP-1 are in the upper part of the panel and closed structures are in the lower part. Arrows (blue and red) indicate the direction and the deviation (length of arrows) of each residue returning to the 3D reference structure of TIMP-1. ( b ) Deformation energy of each TIMP-1 residue (numbering of the mature secreted protein) along mode 7. The lowest deformation energies are coloured blue and the highest are red. ( c ) Visualisation of the TIMP-1 3D-structure of each amino acid deformation energy. Residues with the lowest deformation energy are thin and coloured blue and those with the highest atomic fluctuation are thick and coloured red. ( d ) Cα atomic fluctuation of each TIMP-1 residue (numbering of the mature secreted protein) along mode 7. The lowest atomic fluctuations are coloured blue and the highest are red. ( e ) Visualisation of the TIMP-1 3D-structure of each amino acid atomic fluctuation. Residues with the lowest atomic fluctuations are thin and coloured blue and those with the highest atomic fluctuations are thick and coloured red.
Figure Legend Snippet: Normal mode analysis of TIMP-1 structure. ( a ) Intrinsic dynamic of TIMP-1 along modes 7, 8 and 9. Open structures of TIMP-1 are in the upper part of the panel and closed structures are in the lower part. Arrows (blue and red) indicate the direction and the deviation (length of arrows) of each residue returning to the 3D reference structure of TIMP-1. ( b ) Deformation energy of each TIMP-1 residue (numbering of the mature secreted protein) along mode 7. The lowest deformation energies are coloured blue and the highest are red. ( c ) Visualisation of the TIMP-1 3D-structure of each amino acid deformation energy. Residues with the lowest deformation energy are thin and coloured blue and those with the highest atomic fluctuation are thick and coloured red. ( d ) Cα atomic fluctuation of each TIMP-1 residue (numbering of the mature secreted protein) along mode 7. The lowest atomic fluctuations are coloured blue and the highest are red. ( e ) Visualisation of the TIMP-1 3D-structure of each amino acid atomic fluctuation. Residues with the lowest atomic fluctuations are thin and coloured blue and those with the highest atomic fluctuations are thick and coloured red.

Techniques Used:

Biochemical analysis of LRP-1-mediated endocytosis of wild-type TIMP-1 and mutants. Cortical neurons from mouse embryos cultured for 48 h on poly-L-lysine-coated coverslips were incubated at 4 °C for 1 hour with 5 nM fluoT1-WT, fluoT1-F12A or fluoT1K47A in the presence or absence of 500 nM RAP or 30 µg/mL of blocking LRP-1 polyclonal antibodies (R2629). After careful washing, part of the cells was used to quantify the surface bound and intracellular signal at 4 °C. The other part was then incubated at 37 °C for an additional 10 min to quantify the surface bound and intracellular signal. Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 300 A.U. are considered to be nonspecific. Error bars indicate mean ± SD, *** indicates significantly different with a P-value
Figure Legend Snippet: Biochemical analysis of LRP-1-mediated endocytosis of wild-type TIMP-1 and mutants. Cortical neurons from mouse embryos cultured for 48 h on poly-L-lysine-coated coverslips were incubated at 4 °C for 1 hour with 5 nM fluoT1-WT, fluoT1-F12A or fluoT1K47A in the presence or absence of 500 nM RAP or 30 µg/mL of blocking LRP-1 polyclonal antibodies (R2629). After careful washing, part of the cells was used to quantify the surface bound and intracellular signal at 4 °C. The other part was then incubated at 37 °C for an additional 10 min to quantify the surface bound and intracellular signal. Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 300 A.U. are considered to be nonspecific. Error bars indicate mean ± SD, *** indicates significantly different with a P-value

Techniques Used: Cell Culture, Incubation, Blocking Assay, Fluorescence, Spectrophotometry

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    R&D Systems human timp 1 quantikine elisa kit
    Endogenous <t>TIMP-1</t> interacts with LRP-1 in cortical neurons. 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
    Human Timp 1 Quantikine Elisa Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems quantikine human timp 1 elisa kit
    Immunocytochemistry images for MMP‐1 and <t>TIMP‐1</t> production. MMP‐1 and TIMP‐1 were labelled with fluorescein isothiocyanate (FITC) (green). × 100 magnification. Abbreviations: MMP‐1, matrix metalloproteinase 1; TIMP‐1, tissue inhibitor of metalloproteinases 1
    Quantikine Human Timp 1 Elisa Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantikine human timp 1 elisa kit/product/R&D Systems
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    Endogenous TIMP-1 interacts with LRP-1 in cortical neurons. 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

    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: Endogenous TIMP-1 interacts with LRP-1 in cortical neurons. 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

    Article Snippet: After the resin was collected and washed with tris-buffered saline (TBS), the recombinant proteins were eluted under acidic conditions (0.1 M glycine-HCl, pH 3.5) and the concentrations were determined using the Human TIMP-1 Quantikine ELISA Kit (R & D Systems).

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

    TIMP-1 binding to LRP-1 reduces neurite length. 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

    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: TIMP-1 binding to LRP-1 reduces neurite length. 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

    Article Snippet: After the resin was collected and washed with tris-buffered saline (TBS), the recombinant proteins were eluted under acidic conditions (0.1 M glycine-HCl, pH 3.5) and the concentrations were determined using the Human TIMP-1 Quantikine ELISA Kit (R & D Systems).

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

    TIMP-1 binding to LRP-1 increases growth cone volume. 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

    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: TIMP-1 binding to LRP-1 increases growth cone volume. 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

    Article Snippet: After the resin was collected and washed with tris-buffered saline (TBS), the recombinant proteins were eluted under acidic conditions (0.1 M glycine-HCl, pH 3.5) and the concentrations were determined using the Human TIMP-1 Quantikine ELISA Kit (R & D Systems).

    Techniques: Binding Assay, Blocking Assay, Incubation, Confocal Microscopy, Software

    Inactive T2G mutant of TIMP-1 colocalizes with LRP-1 and exerts similar effects on the morphology of cortical neurons than wild-type TIMP-1. 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

    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: Inactive T2G mutant of TIMP-1 colocalizes with LRP-1 and exerts similar effects on the morphology of cortical neurons than wild-type TIMP-1. 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

    Article Snippet: After the resin was collected and washed with tris-buffered saline (TBS), the recombinant proteins were eluted under acidic conditions (0.1 M glycine-HCl, pH 3.5) and the concentrations were determined using the Human TIMP-1 Quantikine ELISA Kit (R & D Systems).

    Techniques: Mutagenesis, Staining, Confocal Microscopy, Labeling, Software

    Domains II and IV of the extracellular α-chain of LRP-1 are required to bind and promote TIMP-1 endocytosis in CHO cells. 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

    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: Domains II and IV of the extracellular α-chain of LRP-1 are required to bind and promote TIMP-1 endocytosis in CHO cells. 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

    Article Snippet: After the resin was collected and washed with tris-buffered saline (TBS), the recombinant proteins were eluted under acidic conditions (0.1 M glycine-HCl, pH 3.5) and the concentrations were determined using the Human TIMP-1 Quantikine ELISA Kit (R & D Systems).

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

    Liver MPS NASH model demonstrates fibrotic phenotype. PHH alone (Control and Steatosis) or PHH, KC and HSC co-cultures (NASH) were cultured in the MPS platform for 14 days under standard media (control) or high fat conditions (Steatosis and NASH). a Liver microtissues were stained for cytoskeleton (phalloidin), collagen-type I and α-SMA and imaged by confocal microscopy. Representative images shown and scale bars 200 µm. b Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all microtissues within a FOV (min 8, max 12) and two FOV per scaffold. c Secreted fibrosis biomarkers TIMP-1, Pro-collagen 1, YKL-40 and Fibronectin were all measured in cell culture medium by ELISA, at the end of the culture. d The expression of fibrosis genes was analysed in total RNA from steatosis and NASH models using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression levels are expressed as Log10 relative expression compared to housekeeping genes (GAPDH/B2M/HPRT1) and compared between the NASH and steatosis models (red = upregulation, green = downregulation, black = no change, upper line—1.5-fold increase, lower line 1.5-fold decrease). All data points shown with means ± SD highlighted, data generated from a minimum of nine independent cultures (three donors per condition and n = 3 per donor).

    Journal: Communications Biology

    Article Title: Modelling human liver fibrosis in the context of non-alcoholic steatohepatitis using a microphysiological system

    doi: 10.1038/s42003-021-02616-x

    Figure Lengend Snippet: Liver MPS NASH model demonstrates fibrotic phenotype. PHH alone (Control and Steatosis) or PHH, KC and HSC co-cultures (NASH) were cultured in the MPS platform for 14 days under standard media (control) or high fat conditions (Steatosis and NASH). a Liver microtissues were stained for cytoskeleton (phalloidin), collagen-type I and α-SMA and imaged by confocal microscopy. Representative images shown and scale bars 200 µm. b Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all microtissues within a FOV (min 8, max 12) and two FOV per scaffold. c Secreted fibrosis biomarkers TIMP-1, Pro-collagen 1, YKL-40 and Fibronectin were all measured in cell culture medium by ELISA, at the end of the culture. d The expression of fibrosis genes was analysed in total RNA from steatosis and NASH models using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression levels are expressed as Log10 relative expression compared to housekeeping genes (GAPDH/B2M/HPRT1) and compared between the NASH and steatosis models (red = upregulation, green = downregulation, black = no change, upper line—1.5-fold increase, lower line 1.5-fold decrease). All data points shown with means ± SD highlighted, data generated from a minimum of nine independent cultures (three donors per condition and n = 3 per donor).

    Article Snippet: Soluble biomarkers in cell culture medium from the MPS were analysed by enzyme-linked immunosorbent assay (ELISA); IL-6 was measured by Human IL-6 DuoSet ELISA (R & D Systems); MCP-1 was measured by Human CCL2/MCP-1 DuoSet ELISA (R & D Systems); pro-collagen 1 was measured by Human Pro-Collagen I α1 DuoSet (R & D Systems); TIMP-1 was measured by Human TIMP-1 Quantikine ELISA Kit (R & D Systems); Fibronectin was measured by Human Fibronectin DuoSet ELISA (R & D Systems); YKL-40 was measured by Human Chitinase 3-like 1 Quantikine ELISA Kit (R & D Systems); Albumin secretion was measured by Human Albumin AssayMax™ ELISA kit (Assay Pro, St. Charles, MO).

    Techniques: Cell Culture, Staining, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Polymerase Chain Reaction, Generated

    Obeticholic acid and Elafibranor both modulate inflammatory and fibrosis phenotype in liver MPS NASH model and phenotype can additionally be reversed with dietary changes. PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat conditions and dosed with varying concentrations of Obeticholic acid (OCA) and Elafibranor (ELF) QD or vehicle control (control) for 10 days, following an initial 4-day pre-culture phase. a The secreted cytokine profile from liver microtissues was compared by Luminex to determine the effects of each compound on the inflammatory profile of the liver with samples analysed at day 8 and 14 of the culture. Data were normalised by Z-transformation to allow comparison of all analytes and presented as a heatmap. b The expression of fibrosis-associated genes was analysed in total RNA from control, OCA and ELF treated liver microtissues (highest concentration for each compound) using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression changes are expressed as a fold change over control. c Liver microtissues were stained for collagen-type I, α-SMA, nuclei (DAPI - blue), phalloidin (green) and imaged by confocal microscopy. Representative images are shown and scale bars 200 µm. d Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all the microtissues imaged within an MPS scaffold (min 8, max 20 microtissues). e PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat or lean conditions for 30 days. Cultures either remained in the same type of media throughout or were switched at day 15 from fat media to lean media (Fat - > Lean). Cell culture medium samples were analysed for the presence of IL-6, f MCP-1, g TIMP-1, which were measured by ELISA. h Microtissues were stained with Oil-Red O to determine fat loading during culture period, total stain was quantified by absorbance at 510 nm. All datapoints shown, either as box-whisker plots highlighting mean and min-max or with error bars highlighting means ± SD. All data from a minimum of three independent cultures; statistical comparisons made to control samples unless other comparison shown.

    Journal: Communications Biology

    Article Title: Modelling human liver fibrosis in the context of non-alcoholic steatohepatitis using a microphysiological system

    doi: 10.1038/s42003-021-02616-x

    Figure Lengend Snippet: Obeticholic acid and Elafibranor both modulate inflammatory and fibrosis phenotype in liver MPS NASH model and phenotype can additionally be reversed with dietary changes. PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat conditions and dosed with varying concentrations of Obeticholic acid (OCA) and Elafibranor (ELF) QD or vehicle control (control) for 10 days, following an initial 4-day pre-culture phase. a The secreted cytokine profile from liver microtissues was compared by Luminex to determine the effects of each compound on the inflammatory profile of the liver with samples analysed at day 8 and 14 of the culture. Data were normalised by Z-transformation to allow comparison of all analytes and presented as a heatmap. b The expression of fibrosis-associated genes was analysed in total RNA from control, OCA and ELF treated liver microtissues (highest concentration for each compound) using Human Fibrosis RT2 Profiler PCR Arrays. Gene expression changes are expressed as a fold change over control. c Liver microtissues were stained for collagen-type I, α-SMA, nuclei (DAPI - blue), phalloidin (green) and imaged by confocal microscopy. Representative images are shown and scale bars 200 µm. d Staining of microtissue was quantified by measuring total fluorescence intensity throughout individual microtissues, each data point represents an average of all the microtissues imaged within an MPS scaffold (min 8, max 20 microtissues). e PHH, KC and HSC co-cultures were cultured in the MPS platform under high fat or lean conditions for 30 days. Cultures either remained in the same type of media throughout or were switched at day 15 from fat media to lean media (Fat - > Lean). Cell culture medium samples were analysed for the presence of IL-6, f MCP-1, g TIMP-1, which were measured by ELISA. h Microtissues were stained with Oil-Red O to determine fat loading during culture period, total stain was quantified by absorbance at 510 nm. All datapoints shown, either as box-whisker plots highlighting mean and min-max or with error bars highlighting means ± SD. All data from a minimum of three independent cultures; statistical comparisons made to control samples unless other comparison shown.

    Article Snippet: Soluble biomarkers in cell culture medium from the MPS were analysed by enzyme-linked immunosorbent assay (ELISA); IL-6 was measured by Human IL-6 DuoSet ELISA (R & D Systems); MCP-1 was measured by Human CCL2/MCP-1 DuoSet ELISA (R & D Systems); pro-collagen 1 was measured by Human Pro-Collagen I α1 DuoSet (R & D Systems); TIMP-1 was measured by Human TIMP-1 Quantikine ELISA Kit (R & D Systems); Fibronectin was measured by Human Fibronectin DuoSet ELISA (R & D Systems); YKL-40 was measured by Human Chitinase 3-like 1 Quantikine ELISA Kit (R & D Systems); Albumin secretion was measured by Human Albumin AssayMax™ ELISA kit (Assay Pro, St. Charles, MO).

    Techniques: Cell Culture, Luminex, Transformation Assay, Expressing, Concentration Assay, Polymerase Chain Reaction, Staining, Confocal Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay, Whisker Assay

    The results of TIMP-1 (A) and TSP-1 (B) between COPD and COPD-PH group in validation cohort by ELISA. The P -value of each factor was obtained from unpaired Student's t -test analysis. ** P

    Journal: Frontiers in Medicine

    Article Title: TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients

    doi: 10.3389/fmed.2021.774623

    Figure Lengend Snippet: The results of TIMP-1 (A) and TSP-1 (B) between COPD and COPD-PH group in validation cohort by ELISA. The P -value of each factor was obtained from unpaired Student's t -test analysis. ** P

    Article Snippet: Concentrations of Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) and Thrombospondin 1 (TSP-1) in the plasma were measured by Human TIMP-1 Quantikine ELISA Kit (catalog #DTM100, R & D Systems) and Human Thrombospondin-1 Quantikine ELISA Kit (catalog #DTSP10, R & D Systems) according to the manufacturer's instructions.

    Techniques: Enzyme-linked Immunosorbent Assay

    Summary figure of the schematic workflow of the current study. (A) Initial screening of angiogenic factors and cytokines. (B) ELISA verification in validation cohort and related statistical analysis. (C) Involvement of TIMP-1 in the hPASMCs mechanism. COPD, Chronic obstructive pulmonary disease; COPD-PH, Chronic obstructive pulmonary disease associated pulmonary hypertension; CSE, Cigarette smoke exposure; ELISA, Enzyme-Linked Immunosorbent Assay; hPASMCs, Human Pulmonary Artery Smooth Muscle Cells; TIMP-1, Tissue Inhibitor of Metalloproteinases-1. This figure was created using BioRender .

    Journal: Frontiers in Medicine

    Article Title: TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients

    doi: 10.3389/fmed.2021.774623

    Figure Lengend Snippet: Summary figure of the schematic workflow of the current study. (A) Initial screening of angiogenic factors and cytokines. (B) ELISA verification in validation cohort and related statistical analysis. (C) Involvement of TIMP-1 in the hPASMCs mechanism. COPD, Chronic obstructive pulmonary disease; COPD-PH, Chronic obstructive pulmonary disease associated pulmonary hypertension; CSE, Cigarette smoke exposure; ELISA, Enzyme-Linked Immunosorbent Assay; hPASMCs, Human Pulmonary Artery Smooth Muscle Cells; TIMP-1, Tissue Inhibitor of Metalloproteinases-1. This figure was created using BioRender .

    Article Snippet: Concentrations of Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) and Thrombospondin 1 (TSP-1) in the plasma were measured by Human TIMP-1 Quantikine ELISA Kit (catalog #DTM100, R & D Systems) and Human Thrombospondin-1 Quantikine ELISA Kit (catalog #DTSP10, R & D Systems) according to the manufacturer's instructions.

    Techniques: Enzyme-linked Immunosorbent Assay

    Angiogenesis array data. (A) Representative image of angiogenesis array of the plasma from COPD patients without PH [COPD, (a)] and with PH [COPD-PH, (b)] array blots. The levels of angiogenic factors are determined based on their blotting intensity in duplicates. (B–E) Relative protein level of for angiogenesis-associated protein determined by angiogenesis assay (Data were normalized to three pairs of reference spots). (F,G) Based on two-sample independent Student's t -test analysis, TIMP-1 and TSP-1 were significantly higher in COPD-PH than COPD group. Data were present as mean ± SEM ( n = 4). * P

    Journal: Frontiers in Medicine

    Article Title: TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients

    doi: 10.3389/fmed.2021.774623

    Figure Lengend Snippet: Angiogenesis array data. (A) Representative image of angiogenesis array of the plasma from COPD patients without PH [COPD, (a)] and with PH [COPD-PH, (b)] array blots. The levels of angiogenic factors are determined based on their blotting intensity in duplicates. (B–E) Relative protein level of for angiogenesis-associated protein determined by angiogenesis assay (Data were normalized to three pairs of reference spots). (F,G) Based on two-sample independent Student's t -test analysis, TIMP-1 and TSP-1 were significantly higher in COPD-PH than COPD group. Data were present as mean ± SEM ( n = 4). * P

    Article Snippet: Concentrations of Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) and Thrombospondin 1 (TSP-1) in the plasma were measured by Human TIMP-1 Quantikine ELISA Kit (catalog #DTM100, R & D Systems) and Human Thrombospondin-1 Quantikine ELISA Kit (catalog #DTSP10, R & D Systems) according to the manufacturer's instructions.

    Techniques: Angiogenesis Assay

    CSE increased TIMP-1 level in cultured hPASMCs, and TIMP-1 decreased hPASMCs proliferation under CSE. (A) Western Blotting indicates 0.5% CSE treatment for 36 h increased TIMP-1 expression in hPASMCs. (B) TIMP-1 attenuated CSE-stimulated hPASMCs proliferation. CSE, Cigarette Smoking Extract; hPASMCs, human Pulmonary Arterial Smooth Muscle Cell, ** P

    Journal: Frontiers in Medicine

    Article Title: TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients

    doi: 10.3389/fmed.2021.774623

    Figure Lengend Snippet: CSE increased TIMP-1 level in cultured hPASMCs, and TIMP-1 decreased hPASMCs proliferation under CSE. (A) Western Blotting indicates 0.5% CSE treatment for 36 h increased TIMP-1 expression in hPASMCs. (B) TIMP-1 attenuated CSE-stimulated hPASMCs proliferation. CSE, Cigarette Smoking Extract; hPASMCs, human Pulmonary Arterial Smooth Muscle Cell, ** P

    Article Snippet: Concentrations of Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) and Thrombospondin 1 (TSP-1) in the plasma were measured by Human TIMP-1 Quantikine ELISA Kit (catalog #DTM100, R & D Systems) and Human Thrombospondin-1 Quantikine ELISA Kit (catalog #DTSP10, R & D Systems) according to the manufacturer's instructions.

    Techniques: Cell Culture, Western Blot, Expressing

    Immunocytochemistry images for MMP‐1 and TIMP‐1 production. MMP‐1 and TIMP‐1 were labelled with fluorescein isothiocyanate (FITC) (green). × 100 magnification. Abbreviations: MMP‐1, matrix metalloproteinase 1; TIMP‐1, tissue inhibitor of metalloproteinases 1

    Journal: International Wound Journal

    Article Title: Effect of Panax ginseng extract on the activity of diabetic fibroblasts in vitro. Effect of Panax ginseng extract on the activity of diabetic fibroblasts in vitro

    doi: 10.1111/iwj.13091

    Figure Lengend Snippet: Immunocytochemistry images for MMP‐1 and TIMP‐1 production. MMP‐1 and TIMP‐1 were labelled with fluorescein isothiocyanate (FITC) (green). × 100 magnification. Abbreviations: MMP‐1, matrix metalloproteinase 1; TIMP‐1, tissue inhibitor of metalloproteinases 1

    Article Snippet: The levels of human TIMP‐1 were measured in culture media using the Quantikine Human TIMP‐1 ELISA kit (R & D Systems, Minneapolis, Minnesota) according to the manufacturer's instructions.

    Techniques: Immunocytochemistry