active mmp-9 Search Results


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  • 99
    Thermo Fisher mmp9 activities
    Quantification of MMPs in non-malignant breast tissue and tumor homogenates. (A) Homogenates from three representative human breast cancer samples and paired normal tissue, selected from the 25 patients in Fig. 7 were analyzed on a 10% gelatin zymogram. Recombinant active MMP2 and <t>MMP9</t> were used as standards (2 ng per lane). (B) ELISA quantification of six MMPs in five representative human breast cancer samples (red) and paired normal tissue (blue), including the three pairs shown in panel A. Error bars are standard deviations. ND = not detectable.
    Mmp9 Activities, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore active mmp 9
    <t>MMP-9</t> activation of endothelial cells. (A) Effect of stimulation with MMP-9 on HUVEC cell activation (pERK); n = 4. (B) Effect of stimulation with MMP-9 on HUVEC cell apoptosis (caspase-3 activity; p
    Active Mmp 9, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam active mmp 9
    The effect of diabetes on NGAL and <t>MMP-9</t> expression in PVC implant cells and wound fluids at days 3, 6 and 12. (A) NGAL, MMP-9 and MMP-8 mRNA by qRT-PCR, (B) A representative zymogram of wound fluid at day 6, (C-E) Group data for zymography results expressed as Area Under Curve (AUC; determined from band intensity) (C) total MMP-9 (TMMP-9 = proMMP-9 +active MMP-9), (D) active MMP-9, (E) proMMP-9 and (F) NGAL/MMP-9 complex according to molecular weight. Results are from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals and are expressed as Mean ± SEM *P
    Active Mmp 9, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    ANAWA active mmp 9
    Levels of MMP-2, <t>MMP-9,</t> TIMP-1, and TIMP-2 measured by ELISA in the serum of one patient with MPS II. Each bar represents the mean ± SED from experiments performed in duplicate and are presented as % of values of age- and sex-matched
    Active Mmp 9, supplied by ANAWA, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    R&D Systems human active mmp 9
    Effect of mechanical stretch and IL-10 on <t>MMP-9</t> release in epithelial cells and fibroblasts. Release of MMP-9 into the supernatant of E18–19 epithelial cells ( a ) and fibroblasts ( b ) exposed to 20% cyclic stretch for 48 h in the presence or absence
    Human Active Mmp 9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore recombinant active mmp 9
    Photographs from the epicardial surface (top) and endocardial surface (bottom) of hearts from <t>MMP-9</t> gene promoter reporter mice showing regions of positive β-galactosidase staining (dark regions on lighter myocardium) at indicated time points after RF current-induced injury. Positive β-galactosidase staining was observed at 3 d post-RF injury and peaked at 7 d after MI. Scale grid at left represents a square with 2-mm sides.
    Recombinant Active Mmp 9, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam mouse active mmp 9
    R1R2 impairs the invading capacity of fibrocytes through the BM by reducing the proteolytic activity of <t>MMP-9.</t> (A, B) CD45 + , type I collagen + , and CXCR4 + fibrocytes isolated from bleomycin-treated lungs (14 days) were seeded in the upper chamber and treated with R1R2 or the scrambled peptide (1000 nM) for 24 h. CXCL12 (20 ng/mL) or 10% fetal bovine serum (FBS) were added in the bottom wells. CXCL12-induced fibrocyte invasion through Matrigel (A). Data are expressed as relative fluorescence units (RFU). The invasive capacities through Matrigel-coated inserts between R1R2- and scrambled peptide-treated fibrocytes are shown. Results were normalized for FBS-induced fibrocyte invasion. ** P
    Mouse Active Mmp 9, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare active mmp 9
    tPA-Mediated BM Reconstitution Requires Both Plg and MMP Activation Plg +/+ and Plg −/− mice (A–F) and <t>MMP-9</t> +/+ and MMP-9 −/− mice (G–I) were injected with a single dose of 5-FU followed by daily injections with recombinant tPA or carrier. (A) Survival was assessed daily in Plg +/+ (n = 9 per group) and Plg −/− mice (n = 8 per group). WBC counts (B) and BM cell numbers per femur (C) were determined. Error bars represent SEM. (D) BM sections of Plg +/+ mice coinjection with or without tPA were stained for Plg 2 days after 5-FU treatment (brown staining). Plg staining was found along bone-lining cells. (E and F) BM myelogram was performed. Percentage of myeloid (E) and erythroid (F) BM cells are given. (G–I) BM cell numbers per femur (G), WBC counts (H), and survival (I) were assessed in MMP-9 +/+ and MMP-9 −/− mice (n = 12). *p
    Active Mmp 9, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore mouse active mmp 9
    The effect of maternal hypoxia on the activity of MMP-2 and <t>MMP-9.</t> Hearts were isolated from E21 and PD7 rats in the control and hypoxic groups. The activities of MMP-2 and MMP-9 were determined by gelatin zymography. Clear bands indicate positive MMP
    Mouse Active Mmp 9, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology active mmp 9
    <t>MMP-9</t> activation of endothelial cells. (A) Effect of stimulation with MMP-9 on HUVEC cell activation (pERK); n = 4. (B) Effect of stimulation with MMP-9 on HUVEC cell apoptosis (caspase-3 activity; p
    Active Mmp 9, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    AnaSpec active mmp 9
    Age-dependent blood-brain barrier breakdown and elevated cyclophilin A and active <t>MMP-9</t> levels in cerebrospinal fluid (CSF) of cognitively normal APOE4 carriers
    Active Mmp 9, supplied by AnaSpec, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare active mmp 9 elisa kit
    Age-dependent blood-brain barrier breakdown and elevated cyclophilin A and active <t>MMP-9</t> levels in cerebrospinal fluid (CSF) of cognitively normal APOE4 carriers
    Active Mmp 9 Elisa Kit, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    R&D Systems human active mmp 9 kits
    TB patients carrying the two-locus genotype -2518 MCP-1 GG -1607 MMP-1 2G/2G have the highest serum levels of MCP-1 and the highest plasma levels of MMP-1 and MMP9 Values are shown as medians (horizontal lines), the 25th and 75th percentiles (boxes), and ranges (whiskers). Legends in the x-axis mean: 1 = two-locus genotype A/- 1G/-; 2 = two-locus genotype A/- 2G/2G; 3 = two-locus genotype GG 1G/-; 4 = two-locus genotype GG 2G/2G ). Section A: Distribution of serum MCP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the serum levels of MCP-1 across genotypes (ANOVA F = 16.31; p = 0.0001). Section B: Distribution of plasma MMP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-1 across genotypes (ANOVA F = 5.76; p = 0.001). Section C: Distribution of plasma <t>MMP-9</t> values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-9 across genotypes (ANOVA F = 8.25; p = 0.0001). Comparison of means and standard deviations by genotypes are shown at the right of the whiskers and box figures. The p-values are based on the Bonferroni least significant difference test.
    Human Active Mmp 9 Kits, supplied by R&D Systems, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    R&D Systems mature active mmp 9
    GC frass and a TLR2 agonist induced <t>MMP-9</t> release from neutrophils. A. HL-60 cells were treated with PBS, GC frass, Pam-3-Cys (1 μg/ml) with and without pretreatment with lipoprotein lipase (1000U/mg) for 18 h. Supernatants were harvested, clarified,
    Mature Active Mmp 9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare active mmp 9 enzyme
    GC frass and a TLR2 agonist induced <t>MMP-9</t> release from neutrophils. A. HL-60 cells were treated with PBS, GC frass, Pam-3-Cys (1 μg/ml) with and without pretreatment with lipoprotein lipase (1000U/mg) for 18 h. Supernatants were harvested, clarified,
    Active Mmp 9 Enzyme, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    R&D Systems human active mmp 9 fluorokine e kit
    GC frass and a TLR2 agonist induced <t>MMP-9</t> release from neutrophils. A. HL-60 cells were treated with PBS, GC frass, Pam-3-Cys (1 μg/ml) with and without pretreatment with lipoprotein lipase (1000U/mg) for 18 h. Supernatants were harvested, clarified,
    Human Active Mmp 9 Fluorokine E Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 87/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Genmed tissue active mmp 9 fluorescent assay kit
    The binding of CLU with <t>MMP-9</t> or VEGF A. the binding of CLU and MMP-9 or VEGF was determined using immunoprecipitation and Western-blotting. 5-8F cells and 6-10B cells with DNP treatment were immunoprecipitated using anti-CLU body, and MMP-9 or VEGF was detected in the immunocomplexs by Western-blotting (a). MMP-9 or VEGF was respectively immunoprecipitated in the indicated cells using MMP-9 (b) or VEGF (c) antibody, and CLU was detected using Western-blotting. IP: immunoprecipitation; WB: Western-blotting. B. CLU co-localizes and binds with MMP-9. C. CLU co-localizes and binds to VEGF. DNP-treated or untreated 6-10B cell were fixed with paraformaldehyde, stained for CLU (green) and MMP-9 or VEGF (red), and then visualized by immunofluorescence microscopy. The localization and binding of CLU and MMP-9 or VEGF were indicated. Original magnification, ×1000. Scale bar, 50 μm. Arrow, CLU binding to MMP-9 or VEGF.
    Tissue Active Mmp 9 Fluorescent Assay Kit, supplied by Genmed, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    R&D Systems elisa kit fluorokine e human active mmp 9
    Inhibition of <t>MMP-9</t> secretion and activity and intracellular accumulation of MMP-9 in WIN-treated activated primary peripheral monocytes. (a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) using anti-MMP-9 antibodies. The figure shows one representative analysis out of three. WIN inhibited MMP-9 secretion and induced an intracellular accumulation of 92 kDa MMP-9. (b) MMP-9 <t>activity-ELISA</t> of conditioned medium. Upon treatment with 2 and 4 µM WIN, a concentration-dependent reduction of MMP-9-activity was observed. Data are shown as means +/− SD n = 3. **p
    Elisa Kit Fluorokine E Human Active Mmp 9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Prospec recombinant human active mmp 9 catalytic subunit
    Endogenous βig-h3 cleavage by <t>MMP-9</t> in glioma cells. A and B, βig-h3 and MMP-9 in MMP-9-transfected U87MG cells. A, βig-h3 and MMP-9 expression in the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells were examined by immunoblotting. B, MMP-9 activity was detected by gelatin zymography. M is a marker for active MMP-9 and pro-MMP-2 ( n = 3). C, co-immunoprecipitation of MMP-9 with βig-h3 from the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells. After the lysates from the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells were immunoprecipitated ( IP ) with anti-βig-h3 (+) or control rabbit IgG (−), they were subjected to immunoblotting ( IB ) using mouse antibody for Myc ( MMP-9 ) ( n = 4). D, MMP-9 overexpressed in cells alters cell invasion. Cell invasion in vehicle-transfected, MMP-9-transfected, or IL-1β (10 ng/ml)-treated U87MG cells was examined on Matrigels. βig-h3 and MMP-9 proteins from IL-1β-treated human glioma U87MG cells were examined by immunoblotting and zymography. The control represents untreated samples ( n = 3). E, βig-h3 knockdown alters cell invasion. Cell invasion in control siRNA or βig-h3 siRNA-transfected U87MG cells was examined on Matrigels. The βig-h3 and MMP-9 from these cells were examined by immunoblotting and zymography. Control represents control siRNA. (*, p
    Recombinant Human Active Mmp 9 Catalytic Subunit, supplied by Prospec, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mmp2 mmp9 activity
    Marimastat blocks forward neuroblast migration. ( A – C ) Focal administration of the dual <t>MMP-2/MMP-9</t> inhibitor Marimastat (1 µ M/0.1 µL) (Tocris) increased the diameter of the proximal RMS ( B 2 ) but decreased the diameter of the distal
    Mmp2 Mmp9 Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    R&D Systems human active mmp 9 fluorescent assay kit
    Marimastat blocks forward neuroblast migration. ( A – C ) Focal administration of the dual <t>MMP-2/MMP-9</t> inhibitor Marimastat (1 µ M/0.1 µL) (Tocris) increased the diameter of the proximal RMS ( B 2 ) but decreased the diameter of the distal
    Human Active Mmp 9 Fluorescent Assay Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Novus Biologicals anti active mmp 9 antibody
    Marimastat blocks forward neuroblast migration. ( A – C ) Focal administration of the dual <t>MMP-2/MMP-9</t> inhibitor Marimastat (1 µ M/0.1 µL) (Tocris) increased the diameter of the proximal RMS ( B 2 ) but decreased the diameter of the distal
    Anti Active Mmp 9 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Merck KGaA mmp 9 mmp 13 activity
    Marimastat blocks forward neuroblast migration. ( A – C ) Focal administration of the dual <t>MMP-2/MMP-9</t> inhibitor Marimastat (1 µ M/0.1 µL) (Tocris) increased the diameter of the proximal RMS ( B 2 ) but decreased the diameter of the distal
    Mmp 9 Mmp 13 Activity, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore recombinant human mmp 9 pre activated
    Recombinant <t>MMP-9</t> (rMMP-9) induces lobar hemorrhage in brains of C57/BL6 mice (A) Representative images of mouse brains topically treated with or without rMMP-9. C57/BL6 mice at 3 months of age were prepared with a craniotomy followed by careful removal of the dura mater. Pre-activated recombinant MMP-9 (rMMP-9, 0.4 μg) or PBS (control) was topically applied to the surface of mouse brains. Forty eight hours after the treatment, the mice were sacrificed and mouse brains were removed to examine the lobar hemorrhage and imaged by a camera (upper panel) or a low magnification microscope (2.5X, Olympus) (lower panel). (B) Representative H E staining of brain sections from mice topically treated with or without rMMP-9. Mouse brains treated with rMMP-9 or PBS (control) were sectioned and processed for histologic examination by H E staining. Arrows show the vascular rupture in mouse brains treated with rMMP-9. Scale bar = 200 μm.
    Recombinant Human Mmp 9 Pre Activated, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore mmp 9
    Mesenchymal leader cells in the 2D ex vivo injury culture model are enriched for molecules involved in invasion, CD44, MMP-2, and <t>MMP-9.</t> (A) Model of the different regions of the ex vivo mock cataract surgery explant that are separated by microdissection for this study from both top and side views. Cells found in the points of the star-shaped culture that contain the region of the lens that is occupied by epithelial cells in vivo are referred to as the original attachment zone (OAZ, yellow). In response to injury, cells move onto the denuded basement membrane (BM) into the central migration zone (CMZ, purple). (B–D) To examine whether there are migration-specific changes in expression of molecules associated with invasion after wounding, ex vivo injury explants were microdissected into OAZ and CMZ regions and extracted on days 1–3 for Western blot analysis for CD44, MMP-9, MMP-2, and GAPDH (loading control). Graphs depicting Western blot results from four independent studies show the relative expression of CD44, MMP-9, and MMP-2 to GAPDH. CD44, MMP-9, and MMP-2 were predominately expressed in the CMZ region. These molecules decreased in expression as wound healing progressed on day 2 and on day 3, when wound healing is typically completed. MMP-2 and MMP-9 were detected predominantly in their active forms (bottom arrows) compared with their inactive proenzymatic form (top arrows). For both MMP-2 and MMP-9, the active form (lower band) was quantified and is represented on the graphs as a ratio to GAPDH. Immunoblots were quantified using Kodak 1D software, and data were normalized to results at day 1 in the CMZ zone. Values for each graph and relative densities are plotted ±SEM. Representative Western blots are shown below each graph. (E–M) Ex vivo–wounded cultures were fixed and immunostained for vimentin (F, I, L; green) and colabeled for CD44 (E, G; red), MMP-9 (H, J; red), or MMP-2 (K, M; red). Vimentin-rich mesenchymal cells at the leading edge were enriched for MMP-2, MMP-9, and CD44. CD44 localized at cell–cell interfaces and to the tips of these cells at the leading edge (E–G, arrow). MMP-9 strongly localized to vimentin-rich cells, sometimes colocalized with vimentin (H–J, arrow) and also to regions in the area to be repaired just beyond the cells (H, J, arrowhead). Bar, 10 μm. (N) The role of MMP-2/9 function on wound healing in the 2D ex vivo injury cultures was evaluated using inhibitors to MMP-2, MMP-9, or MMP-2/9. Phase images were taken on days 0–3, from which the open wound area was calculated in at least six capsules and quantified using NIS Elements analysis software. Open wound area was plotted on the graphs ±SEM. Inhibition of MMP-2 and/or MMP-9 function resulted in only a slight delay in wound closure.
    Mmp 9, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 3214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc mmp 9
    Tan-IIA inhibited migratory and invasive ability in human BCa cells. ( A ) Human BCa cells were treated with 0.2% DMSO as a vehicle control or 4 μg/mL Tan-IIA for 24 h and then seeded onto the transwell hanging insert for migration (24 h) and invasion (48 h) assays. Images were captured using an inverted microscope with 200× magnification; Scale bar: 50 μm. The migration and invasion of BCa cells were quantified by counting the stained cells that migrated into the underside of the hanging insert membrane; ( B ) human BCa cells were treated with different concentrations of Tan-IIA (1, 2 and 4 μg/mL) for 48 h. The protein of total cell lysates were then used to detect <t>MMP-9/-2</t> protein expression using western blot, and the ( C ) supernatant was used to detect the enzymatic activity using zymography analysis. M: marker. Data are presented as means ± S.D. from three different experiments. ** p
    Mmp 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare mmp 9 activity assays
    HPV-16 E7 up-regulates <t>MMP-9</t> activity in organotypic cultures. Primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. Cells were then differentiated in organotypic cultures for 9 to 11 days. A, Determination of gelatinase activity in organotypic cultures homogenates (epidermis separated from dermis). Equal amounts of proteins were loaded. Note the increase in MMP-9 gelatinase activity in HPV-16 E7wt and E6E7- expressing epidermis. B-B′, densitometry of pro-MMP-9 bands in epidermis and dermis, respectively. C, MMP-9 levels in epidermis homogenates were determined by Western blot. P
    Mmp 9 Activity Assays, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam active human mmp 9 full length protein
    Faecal <t>MMP-9</t> levels in adenomas.
    Active Human Mmp 9 Full Length Protein, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics mmp 9 activities
    A model proposed for the cross-talk between N-cadherin cleavage and <t>MMP-9</t> expression in NPC cell invasion
    Mmp 9 Activities, supplied by Epitomics, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Quickzyme mmp 9 activity assays
    TXA reduces activation of MMP-2 and <t>MMP-9</t> in the injured spinal cord. Contusion SCI was induced by the Infinite Horizons impactor in C57BL/6 mice treated without or with TXA. Mice were treated with a bolus intravenous injection of TXA just after SCI followed by per os administration of TXA (20 mg/mL of drinking water). The concentrations of active MMP-2 ( a ) and MMP-9 ( b ) in the spinal cord were assessed at day 0 (laminectomised mice without SCI; Sham), day 1 (1 dpi), or day 7 (7 dpi). Values and error bars represent mean ± SD ( n = 6 in each group). * P
    Mmp 9 Activity Assays, supplied by Quickzyme, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Quantification of MMPs in non-malignant breast tissue and tumor homogenates. (A) Homogenates from three representative human breast cancer samples and paired normal tissue, selected from the 25 patients in Fig. 7 were analyzed on a 10% gelatin zymogram. Recombinant active MMP2 and MMP9 were used as standards (2 ng per lane). (B) ELISA quantification of six MMPs in five representative human breast cancer samples (red) and paired normal tissue (blue), including the three pairs shown in panel A. Error bars are standard deviations. ND = not detectable.

    Journal: Theranostics

    Article Title: Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620

    doi: 10.7150/thno.20678

    Figure Lengend Snippet: Quantification of MMPs in non-malignant breast tissue and tumor homogenates. (A) Homogenates from three representative human breast cancer samples and paired normal tissue, selected from the 25 patients in Fig. 7 were analyzed on a 10% gelatin zymogram. Recombinant active MMP2 and MMP9 were used as standards (2 ng per lane). (B) ELISA quantification of six MMPs in five representative human breast cancer samples (red) and paired normal tissue (blue), including the three pairs shown in panel A. Error bars are standard deviations. ND = not detectable.

    Article Snippet: MMP2 and MMP9 activities were visualized in the tissue homogenates using Novex gelatin 10 % zymogram gels (Life Technologies, Carlsbad, CA) according to the recommended protocol.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

    AVB-620 structure, absorbance and fluorescence properties. (A) Structure of AVB-620. (B) Absorbance spectrum of 17.5 µg/mL AVB-620 in 80/20 PBS/acetonitrile from 500-800 nm. (C) AVB-620 fluorescence emission spectrum in TCNB buffer using 630 nm excitation and an emission wavelength range of 620-850 nm (red trace). Addition of MMP9 hydrolyzes AVB-620, disrupting FRET between Cy5 and Cy7 dyes, and results in large fluorescence change (blue trace). Representative spectra are shown from six experiments.

    Journal: Theranostics

    Article Title: Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620

    doi: 10.7150/thno.20678

    Figure Lengend Snippet: AVB-620 structure, absorbance and fluorescence properties. (A) Structure of AVB-620. (B) Absorbance spectrum of 17.5 µg/mL AVB-620 in 80/20 PBS/acetonitrile from 500-800 nm. (C) AVB-620 fluorescence emission spectrum in TCNB buffer using 630 nm excitation and an emission wavelength range of 620-850 nm (red trace). Addition of MMP9 hydrolyzes AVB-620, disrupting FRET between Cy5 and Cy7 dyes, and results in large fluorescence change (blue trace). Representative spectra are shown from six experiments.

    Article Snippet: MMP2 and MMP9 activities were visualized in the tissue homogenates using Novex gelatin 10 % zymogram gels (Life Technologies, Carlsbad, CA) according to the recommended protocol.

    Techniques: Fluorescence

    Profiles of MMP cleavage of AVB-620. (A) Catalytic efficiency of a panel of MMPs for cleavage of AVB-620. (B) Catalytic efficiency of human MMP2 and MMP9 versus mouse MMP2 and MMP9. Error bars are standard deviations. ND = not detectable.

    Journal: Theranostics

    Article Title: Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620

    doi: 10.7150/thno.20678

    Figure Lengend Snippet: Profiles of MMP cleavage of AVB-620. (A) Catalytic efficiency of a panel of MMPs for cleavage of AVB-620. (B) Catalytic efficiency of human MMP2 and MMP9 versus mouse MMP2 and MMP9. Error bars are standard deviations. ND = not detectable.

    Article Snippet: MMP2 and MMP9 activities were visualized in the tissue homogenates using Novex gelatin 10 % zymogram gels (Life Technologies, Carlsbad, CA) according to the recommended protocol.

    Techniques:

    Activation of IL-33-ST2-NF-κB-MMP9 signaling promotes metastasis. ( A ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing WT, Il33 –/– , or St2 –/– mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule (T) and surrounding lung tissues (L) (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( B ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or soluble ST2 (sST2) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( C ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving PBS or clodronate treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( D ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or DHMEQ (a NF-κB inhibitor) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( E ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or SB-3CT (a MMP9 inhibitor) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( F ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing WT or Mmp9 –/– mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group).

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: Activation of IL-33-ST2-NF-κB-MMP9 signaling promotes metastasis. ( A ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing WT, Il33 –/– , or St2 –/– mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule (T) and surrounding lung tissues (L) (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( B ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or soluble ST2 (sST2) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( C ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving PBS or clodronate treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( D ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or DHMEQ (a NF-κB inhibitor) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( E ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing mice receiving vehicle or SB-3CT (a MMP9 inhibitor) treatments. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( F ) Visible surface pulmonary metastatic nodules and lung histology of Panc02 tumor-bearing WT or Mmp9 –/– mice. Arrowheads indicate lung surface metastatic nodules. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group).

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Activation Assay, Mouse Assay

    Mechanisms of IL-33–mediated crosstalk between CAFs and TAMs in cancer metastasis. Mechanistic insights on IL-33–mediated crosstalk between pericytes (PCs)/cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in cancer metastasis. PDGFRβ + PCs/CAFs produce high levels of IL-33, which recruits TAMs and induces M2 macrophage polarization through its ST2 receptor. IL-33–stimulated TAMs produce high levels of MMP9 through activation of NF-κB, which transcriptionally activates the MMP9 promoter. MMP9 in turn degrades laminin, one of the key components in the basement membrane (BM). Deterioration of BM around the tumor vasculature allows tumor cells (TCs) to intravasate into the circulation. Circulating tumor cells (CTCs) eventually recolonize in distal organs, such as lungs, for further growth to become the clinically detectable metastatic mass.

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: Mechanisms of IL-33–mediated crosstalk between CAFs and TAMs in cancer metastasis. Mechanistic insights on IL-33–mediated crosstalk between pericytes (PCs)/cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in cancer metastasis. PDGFRβ + PCs/CAFs produce high levels of IL-33, which recruits TAMs and induces M2 macrophage polarization through its ST2 receptor. IL-33–stimulated TAMs produce high levels of MMP9 through activation of NF-κB, which transcriptionally activates the MMP9 promoter. MMP9 in turn degrades laminin, one of the key components in the basement membrane (BM). Deterioration of BM around the tumor vasculature allows tumor cells (TCs) to intravasate into the circulation. Circulating tumor cells (CTCs) eventually recolonize in distal organs, such as lungs, for further growth to become the clinically detectable metastatic mass.

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Activation Assay

    IL-33–induced MMP9 in macrophages promotes metastasis in human PDAC. ( A ) IL-33 protein levels in human melanoma and pancreatic cancers ( n = 3 samples per group). ( B ) Staining of MiaPaCa2 PDAC and UACC257 melanoma tumor tissues. Light blue indicates fibrotic components (Masson’s trichrome; scale bar: 50 μm). ( C ) qPCR quantification of Mmp9 mRNA expression in 3 human pancreatic tumors ( n = 6 samples per group). ( D ) Immunohistochemical staining and quantification of Iba1 + (red) macrophages in vehicle- and a soluble ST2–treated MiaPaCa2 tumors ( n = 8 random fields per group; scale bar: 100 μm). ( E ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in vehicle- and a soluble ST2–treated MiaPaCa2 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( F ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in vehicle- and SB-3CT–treated MiaPaCa2 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( G ) Lung histology of MiaPaCa2 tumor-bearing mice receiving vehicle or a soluble ST2 treatments. Dashed line marks the border between the metastatic nodule (T) and surrounding lung tissues (L) (scale bar: 250 μm). Quantification of percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( H ) Lung histology of MiaPaCa2 tumor-bearing mice receiving vehicle or SB-3CT treatments. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). Mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: IL-33–induced MMP9 in macrophages promotes metastasis in human PDAC. ( A ) IL-33 protein levels in human melanoma and pancreatic cancers ( n = 3 samples per group). ( B ) Staining of MiaPaCa2 PDAC and UACC257 melanoma tumor tissues. Light blue indicates fibrotic components (Masson’s trichrome; scale bar: 50 μm). ( C ) qPCR quantification of Mmp9 mRNA expression in 3 human pancreatic tumors ( n = 6 samples per group). ( D ) Immunohistochemical staining and quantification of Iba1 + (red) macrophages in vehicle- and a soluble ST2–treated MiaPaCa2 tumors ( n = 8 random fields per group; scale bar: 100 μm). ( E ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in vehicle- and a soluble ST2–treated MiaPaCa2 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( F ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in vehicle- and SB-3CT–treated MiaPaCa2 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( G ) Lung histology of MiaPaCa2 tumor-bearing mice receiving vehicle or a soluble ST2 treatments. Dashed line marks the border between the metastatic nodule (T) and surrounding lung tissues (L) (scale bar: 250 μm). Quantification of percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). ( H ) Lung histology of MiaPaCa2 tumor-bearing mice receiving vehicle or SB-3CT treatments. Dashed line marks the border between the metastatic nodule and surrounding lung tissues (scale bar: 250 μm). Quantification of the percentage of animals with visible pulmonary metastasis ( n = 6–10 mice per group). Mean ± SEM. * P

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry, Mouse Assay

    IL-33 upregulates MMP9 in macrophages through ST2 activation. ( A ) Heatmap of a subset of cancer metastasis–related genes by genome-wide expression profiling of IL-33–stimulated macrophages ( n = 3 samples per group). ( B ) qPCR quantification of mRNA expression levels of Mmps in IL-33–stimulated macrophages ( n = 6 samples per group). ( C ) Inhibition of Mmp9 mRNA expression of IL-33–stimulated macrophages by a soluble ST2 (IL-33 trap) ( n = 6 samples per group). sST2, soluble ST2. ( D ) ELISA quantification of active MMP9 protein in the conditional medium from IL-33–stimulated macrophages ( n = 3 samples per group). ( E ) qPCR quantification of Mmp9 mRNA levels in F4/80 + cells isolated from Panc02 tumors grown in WT or St2 –/– mice ( n = 6 samples per group). ( F ) Detection of MMP9 protease activity by a gelatin-based zymography assay in conditional medium derived from IL-33–stimulated macrophages in the presence or absence of a soluble ST2 ( n = 3 samples per group). NT, nontreated. Mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: IL-33 upregulates MMP9 in macrophages through ST2 activation. ( A ) Heatmap of a subset of cancer metastasis–related genes by genome-wide expression profiling of IL-33–stimulated macrophages ( n = 3 samples per group). ( B ) qPCR quantification of mRNA expression levels of Mmps in IL-33–stimulated macrophages ( n = 6 samples per group). ( C ) Inhibition of Mmp9 mRNA expression of IL-33–stimulated macrophages by a soluble ST2 (IL-33 trap) ( n = 6 samples per group). sST2, soluble ST2. ( D ) ELISA quantification of active MMP9 protein in the conditional medium from IL-33–stimulated macrophages ( n = 3 samples per group). ( E ) qPCR quantification of Mmp9 mRNA levels in F4/80 + cells isolated from Panc02 tumors grown in WT or St2 –/– mice ( n = 6 samples per group). ( F ) Detection of MMP9 protease activity by a gelatin-based zymography assay in conditional medium derived from IL-33–stimulated macrophages in the presence or absence of a soluble ST2 ( n = 3 samples per group). NT, nontreated. Mean ± SEM. * P

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Activation Assay, Genome Wide, Expressing, Real-time Polymerase Chain Reaction, Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Mouse Assay, Activity Assay, Zymography Assay, Derivative Assay

    Transcriptional regulation of the Mmp9 promoter activity by IL-33- NF-κB signaling. ( A ) Western immunoblot analysis of phosphorylation of IκBα, Erk, and p38 in vehicle- and IL-33–treated macrophages in the presence or absence of their specific inhibitors. β-Actin served as a loading control ( n = 3 samples per group). WA, withaferin A; SB, SB203580. ( B ) qPCR quantification of Mmp9 mRNA levels of IL-33–stimulated macrophages in the presence or absence of their specific inhibitors ( n = 6 samples per group). ( C ) Western immunoblot analysis of phosphorylation of IκBα in IL-33–stimulated macrophages in the presence or absence of a soluble ST2 (sST2). β-Actin served as a loading control ( n = 3 samples per group). ( D ) A network scheme demonstrating protein-protein interactions linking IL-33 to NF-κB. Irak, IL-1 receptor–associated kinase; MyD88, myeloid differentiation primary response 88. ( E ) Immunostaining of NF-κB (green) in IL-33–stimulated macrophages at 5-, 30-, or 60-minute time points. Cell nuclei were counterstained with DAPI (red; scale bar: 25 μm). Overlapping yellow signals were randomly quantified ( n = 3 fields per group). ( F ) Immunostaining of NF-κB (green) in IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A. Cell nuclei were counterstained with DAPI (red; scale bar: 50 μm). Overlapping yellow signals were quantified ( n = 4 random fields per group). ( G ) Detection of MMP9 protease activity by a gelatin-based zymography assay in conditional medium derived from IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A ( n = 4 samples per group). ( H ) qPCR quantification of Mmp9 mRNA levels in IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A ( n = 6 samples per group). ( I ) Schematic indicating 5 NF-κB–binding sites located in the proximal region of the mouse Mmp9 promoter. ( J ) Mmp9 promoter activity in macrophages by the luciferase reporter assay. Relative luciferase activity was measured after IL-33 stimulation in the presence or absence of different concentrations of withaferin A ( n = 3 samples per group). ( K ) Schematic showing the IL-33-ST2-NF-κB-MMP9 signaling pathway. IKK, IκB kinase. Mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: Transcriptional regulation of the Mmp9 promoter activity by IL-33- NF-κB signaling. ( A ) Western immunoblot analysis of phosphorylation of IκBα, Erk, and p38 in vehicle- and IL-33–treated macrophages in the presence or absence of their specific inhibitors. β-Actin served as a loading control ( n = 3 samples per group). WA, withaferin A; SB, SB203580. ( B ) qPCR quantification of Mmp9 mRNA levels of IL-33–stimulated macrophages in the presence or absence of their specific inhibitors ( n = 6 samples per group). ( C ) Western immunoblot analysis of phosphorylation of IκBα in IL-33–stimulated macrophages in the presence or absence of a soluble ST2 (sST2). β-Actin served as a loading control ( n = 3 samples per group). ( D ) A network scheme demonstrating protein-protein interactions linking IL-33 to NF-κB. Irak, IL-1 receptor–associated kinase; MyD88, myeloid differentiation primary response 88. ( E ) Immunostaining of NF-κB (green) in IL-33–stimulated macrophages at 5-, 30-, or 60-minute time points. Cell nuclei were counterstained with DAPI (red; scale bar: 25 μm). Overlapping yellow signals were randomly quantified ( n = 3 fields per group). ( F ) Immunostaining of NF-κB (green) in IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A. Cell nuclei were counterstained with DAPI (red; scale bar: 50 μm). Overlapping yellow signals were quantified ( n = 4 random fields per group). ( G ) Detection of MMP9 protease activity by a gelatin-based zymography assay in conditional medium derived from IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A ( n = 4 samples per group). ( H ) qPCR quantification of Mmp9 mRNA levels in IL-33–stimulated macrophages in the presence or absence of different concentrations of withaferin A ( n = 6 samples per group). ( I ) Schematic indicating 5 NF-κB–binding sites located in the proximal region of the mouse Mmp9 promoter. ( J ) Mmp9 promoter activity in macrophages by the luciferase reporter assay. Relative luciferase activity was measured after IL-33 stimulation in the presence or absence of different concentrations of withaferin A ( n = 3 samples per group). ( K ) Schematic showing the IL-33-ST2-NF-κB-MMP9 signaling pathway. IKK, IκB kinase. Mean ± SEM. * P

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Immunostaining, Zymography Assay, Derivative Assay, Binding Assay, Luciferase, Reporter Assay

    IL-33-ST2–dependent production of MMP9 in macrophages. ( A ) Immunohistochemical staining and quantification of Iba1 + (red) and CD206 + (green) macrophages in Panc02 tumors grown in WT, Il33 –/– , or St2 –/– mice ( n = 8 random fields per group; scale bar: 100 μm). ( B ) Immunohistochemical staining and quantification of Iba1 + (red) and CD206 + (green) macrophages in vehicle- and soluble ST2–treated (sST2-treated) Panc02 tumors ( n = 8 random fields per group; scale bar: 100 μm). ( C ) qPCR quantification of Mmp9 mRNA levels in Panc02 tumors grown in WT, Il33 –/– , or St2 –/– mice or WT mice treated with a soluble ST2 ( n = 6 samples per group). ( D ) qPCR quantification of Mmp9 mRNA levels of PBS- and clodronate-treated Panc02 tumors ( n = 6 samples per group). ( E ) qPCR quantification of Mmp9 mRNA levels in Panc02 tumors treated with vehicle and DHMEQ (a NF-κB inhibitor) ( n = 6 samples per group). ( F ) Detection of MMP9 protease activity by a gelatin-based zymography assay in PBS- and clodronate-treated Panc02 tumors ( n = 4 samples per group). ( G ) ELISA quantification of active MMP9 protein in PBS- and clodronate-treated Panc02 tumors ( n = 3 samples per group). Mean ± SEM. ** P

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: IL-33-ST2–dependent production of MMP9 in macrophages. ( A ) Immunohistochemical staining and quantification of Iba1 + (red) and CD206 + (green) macrophages in Panc02 tumors grown in WT, Il33 –/– , or St2 –/– mice ( n = 8 random fields per group; scale bar: 100 μm). ( B ) Immunohistochemical staining and quantification of Iba1 + (red) and CD206 + (green) macrophages in vehicle- and soluble ST2–treated (sST2-treated) Panc02 tumors ( n = 8 random fields per group; scale bar: 100 μm). ( C ) qPCR quantification of Mmp9 mRNA levels in Panc02 tumors grown in WT, Il33 –/– , or St2 –/– mice or WT mice treated with a soluble ST2 ( n = 6 samples per group). ( D ) qPCR quantification of Mmp9 mRNA levels of PBS- and clodronate-treated Panc02 tumors ( n = 6 samples per group). ( E ) qPCR quantification of Mmp9 mRNA levels in Panc02 tumors treated with vehicle and DHMEQ (a NF-κB inhibitor) ( n = 6 samples per group). ( F ) Detection of MMP9 protease activity by a gelatin-based zymography assay in PBS- and clodronate-treated Panc02 tumors ( n = 4 samples per group). ( G ) ELISA quantification of active MMP9 protein in PBS- and clodronate-treated Panc02 tumors ( n = 3 samples per group). Mean ± SEM. ** P

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Real-time Polymerase Chain Reaction, Activity Assay, Zymography Assay, Enzyme-linked Immunosorbent Assay

    Degradation of the basement membrane by the IL-33-ST2-NF-κB-MMP9 axis. ( A ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors grown in WT, Il33 –/– , or St2 –/– mice. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( B ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in vehicle- or soluble ST2–treated Panc02 tumors Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( C ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in PBS- or clodronate-treated Panc02 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( D ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors treated with vehicle or DHMEQ (a NF-κB inhibitor). Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( E ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors treated with vehicle or SB-3CT (a MMP9 inhibitor). Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( F ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors grown in WT or Mmp9 –/– mice. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). Mean ± SEM. ** P

    Journal: JCI Insight

    Article Title: Molecular mechanisms of IL-33–mediated stromal interactions in cancer metastasis

    doi: 10.1172/jci.insight.122375

    Figure Lengend Snippet: Degradation of the basement membrane by the IL-33-ST2-NF-κB-MMP9 axis. ( A ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors grown in WT, Il33 –/– , or St2 –/– mice. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( B ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in vehicle- or soluble ST2–treated Panc02 tumors Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( C ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in PBS- or clodronate-treated Panc02 tumors. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( D ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors treated with vehicle or DHMEQ (a NF-κB inhibitor). Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( E ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors treated with vehicle or SB-3CT (a MMP9 inhibitor). Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). ( F ) Immunohistochemical staining and quantification of CD31 + (red) and laminin + (green) structures in Panc02 tumors grown in WT or Mmp9 –/– mice. Arrowheads indicate laminin/CD31 double-positive signals. Quantification of the percentage of laminin + /CD31 + signals per field ( n = 8 random fields per group; scale bar: 100 μm). Mean ± SEM. ** P

    Article Snippet: Total tissue homogenates or conditional media were collected to measure MMP9 activity using a zymography-based assay (Zymography, Invitrogen).

    Techniques: Immunohistochemistry, Staining, Mouse Assay

    Proposed Ingenuity model describing the relationship between MMPs and TIMPs. Mmp1 and Mmp13 can induce Mmp9 transcription, and Timp3 can inhibit Mmp9 . An increase in Mmp9 can activate major signaling pathways like NFκB and mitogen-activated protein

    Journal: Carcinogenesis

    Article Title: Tpl2 knockout keratinocytes have increased biomarkers for invasion and metastasis

    doi: 10.1093/carcin/bgt319

    Figure Lengend Snippet: Proposed Ingenuity model describing the relationship between MMPs and TIMPs. Mmp1 and Mmp13 can induce Mmp9 transcription, and Timp3 can inhibit Mmp9 . An increase in Mmp9 can activate major signaling pathways like NFκB and mitogen-activated protein

    Article Snippet: To measure Mmp2 and Mmp9 enzymatic activity, we performed zymography using 10% Tris-Glycine gels containing 0.1% gelatin (Invitrogen).

    Techniques:

    Tpl2 −/− primary keratinocytes have elevated gene expression of MMPs, reduced expression of Timp inhibitors and increased levels of active Mmp9 in vivo and in vitro . ( A ) qPCR results for Mmp1 , 2 , 9 and 13 and Timp1 , 2 , 3 and 4 . Total

    Journal: Carcinogenesis

    Article Title: Tpl2 knockout keratinocytes have increased biomarkers for invasion and metastasis

    doi: 10.1093/carcin/bgt319

    Figure Lengend Snippet: Tpl2 −/− primary keratinocytes have elevated gene expression of MMPs, reduced expression of Timp inhibitors and increased levels of active Mmp9 in vivo and in vitro . ( A ) qPCR results for Mmp1 , 2 , 9 and 13 and Timp1 , 2 , 3 and 4 . Total

    Article Snippet: To measure Mmp2 and Mmp9 enzymatic activity, we performed zymography using 10% Tris-Glycine gels containing 0.1% gelatin (Invitrogen).

    Techniques: Expressing, In Vivo, In Vitro, Real-time Polymerase Chain Reaction

    a Showing Western Blot band of Cx43 with treatment of hypoxia in the absence or presence of JSH-23 (30 μM) or SP600125 (10 μM) at 6 h and 12 h. b–f The quantified results are depicted in the form of bar graphs. Cx43 protein expression with treatment for 6 h ( b ) and 12 h ( c ). Hypoxia induced a decrease in Cx43 expression, however, this effect was notably attenuated by both NF-κB and AP-1/c-Jun inhibitors, JSH-23 and SP600125, respectively. The ratio of P-Cx43 to T-Cx43 is shown in ( d–e ). MMP-9 promoter activity assessed with Dual-Luciferase Reporter Assay System was shown in f . Results represent the mean ± SEM of three separate experiments. ( Asterisk ) Significantly different as compared to the control. ( Hash ) data in the groups co-treated with hypoxia and specific inhibitor are significantly different from those treated with hypoxia alone

    Journal: Molecular and Cellular Biochemistry

    Article Title: Hypoxia induces connexin 43 dysregulation by modulating matrix metalloproteinases via MAPK signaling

    doi: 10.1007/s11010-013-1793-5

    Figure Lengend Snippet: a Showing Western Blot band of Cx43 with treatment of hypoxia in the absence or presence of JSH-23 (30 μM) or SP600125 (10 μM) at 6 h and 12 h. b–f The quantified results are depicted in the form of bar graphs. Cx43 protein expression with treatment for 6 h ( b ) and 12 h ( c ). Hypoxia induced a decrease in Cx43 expression, however, this effect was notably attenuated by both NF-κB and AP-1/c-Jun inhibitors, JSH-23 and SP600125, respectively. The ratio of P-Cx43 to T-Cx43 is shown in ( d–e ). MMP-9 promoter activity assessed with Dual-Luciferase Reporter Assay System was shown in f . Results represent the mean ± SEM of three separate experiments. ( Asterisk ) Significantly different as compared to the control. ( Hash ) data in the groups co-treated with hypoxia and specific inhibitor are significantly different from those treated with hypoxia alone

    Article Snippet: MMP-9 activity assay MMP-9 activity was assessed by gelatin zymography [ ] using premade 10 % polyacrylamide gels containing 0.1 % gelatin and 10 μL serum-free media from the treated cultures; these procedures were performed according to the instructions provided by the manufacturer (Invitrogen).

    Techniques: Western Blot, Expressing, Activity Assay, Luciferase, Reporter Assay

    Schematic diagram of the MAPK signaling pathways involved in regulation of Cx43 expression by modulating MMP-9 enzyme. The regulation of Cx43 expression was studied by blocking the pathways of PI3K/Akt or MEK/ERK 1/2 NF-κB or AP-1/c-Jun and MMPs, respectively, with specific inhibitors. The signaling cascades, transcription factors NF-κB and AP-1/c-Jun, and MMPs activity appear to be critical factors in hypoxia-induced disruption of gap junction integrity

    Journal: Molecular and Cellular Biochemistry

    Article Title: Hypoxia induces connexin 43 dysregulation by modulating matrix metalloproteinases via MAPK signaling

    doi: 10.1007/s11010-013-1793-5

    Figure Lengend Snippet: Schematic diagram of the MAPK signaling pathways involved in regulation of Cx43 expression by modulating MMP-9 enzyme. The regulation of Cx43 expression was studied by blocking the pathways of PI3K/Akt or MEK/ERK 1/2 NF-κB or AP-1/c-Jun and MMPs, respectively, with specific inhibitors. The signaling cascades, transcription factors NF-κB and AP-1/c-Jun, and MMPs activity appear to be critical factors in hypoxia-induced disruption of gap junction integrity

    Article Snippet: MMP-9 activity assay MMP-9 activity was assessed by gelatin zymography [ ] using premade 10 % polyacrylamide gels containing 0.1 % gelatin and 10 μL serum-free media from the treated cultures; these procedures were performed according to the instructions provided by the manufacturer (Invitrogen).

    Techniques: Expressing, Blocking Assay, Activity Assay

    Effect EEOS on the expression of uPA, uPAR and EGFR in NCI-H460 cells. (A) RT-PCR analysis showed that EEOS attenuated the expression of uPA, uPAR and EGFR in NCI-H460 cells. (B) Effect EEOS on the mRNA expression of uPA in OPN treated NCI-H460 cells. (C) Effect EEOS on MMP-9 activity in NCI-H460 cells by ELISA.

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Inhibitory effect of ethanol extract of Ocimum sanctum on osteopontin mediated metastasis of NCI-H460 non-small cell lung cancer cells

    doi: 10.1186/1472-6882-14-419

    Figure Lengend Snippet: Effect EEOS on the expression of uPA, uPAR and EGFR in NCI-H460 cells. (A) RT-PCR analysis showed that EEOS attenuated the expression of uPA, uPAR and EGFR in NCI-H460 cells. (B) Effect EEOS on the mRNA expression of uPA in OPN treated NCI-H460 cells. (C) Effect EEOS on MMP-9 activity in NCI-H460 cells by ELISA.

    Article Snippet: MMP-9 enzymatic activities were assayed by ELISA Kit for MMP-9 (Invitrogen, Camarillo, CA, USA) [ ].

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Enzyme-linked Immunosorbent Assay

    Effect of PI3K inhibitor LY294002 on PI3K signaling and MMP-9 activity in OPN treated NCI-H460 cells. (A) Effect of EEOS and LY294002 on PI3K/Akt signaling in OPN treated NCI-H460. (B) Effect of EEOS and LY294002 on uPA expression in OPN treated NCI-H460 by RT-PCR. (C) Effect of EEOS and LY294002 on MMP-9 activity in OPN treated NCI-H460 by ELISA. Values represent means ± S.D. *P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Inhibitory effect of ethanol extract of Ocimum sanctum on osteopontin mediated metastasis of NCI-H460 non-small cell lung cancer cells

    doi: 10.1186/1472-6882-14-419

    Figure Lengend Snippet: Effect of PI3K inhibitor LY294002 on PI3K signaling and MMP-9 activity in OPN treated NCI-H460 cells. (A) Effect of EEOS and LY294002 on PI3K/Akt signaling in OPN treated NCI-H460. (B) Effect of EEOS and LY294002 on uPA expression in OPN treated NCI-H460 by RT-PCR. (C) Effect of EEOS and LY294002 on MMP-9 activity in OPN treated NCI-H460 by ELISA. Values represent means ± S.D. *P

    Article Snippet: MMP-9 enzymatic activities were assayed by ELISA Kit for MMP-9 (Invitrogen, Camarillo, CA, USA) [ ].

    Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effects of particle exposure on IL-12, MMP-2, MMP-9 and TIMP-1 levels in the 1st BALF. The first acellular BALF was isolated from control and particle-exposed animals at 1- and 28-day post exposure. Time-dependent effects of particle induced inflammatory

    Journal: Toxicology and applied pharmacology

    Article Title: Effects of amorphous silica coating on cerium oxide nanoparticles induced pulmonary responses

    doi: 10.1016/j.taap.2015.07.012

    Figure Lengend Snippet: Effects of particle exposure on IL-12, MMP-2, MMP-9 and TIMP-1 levels in the 1st BALF. The first acellular BALF was isolated from control and particle-exposed animals at 1- and 28-day post exposure. Time-dependent effects of particle induced inflammatory

    Article Snippet: To determine the MMP-9 activity in the first acellular BALF, 15 μg of acellullar BALF protein was loaded onto 10% Novex Zymogram (Gelatinase) gels, consisting of a 10% Tris-Glycine gel with 0.1% gelatin as the substrate (Life Technologies; Grand Island, NY), according to the manufacturer’s instructions.

    Techniques: Isolation

    Histopathologic examination of PC3 bone tumors Mice were killed on day 50 after tumor cell implantation, and tumor-bearing legs were harvested and evaluated. Representative photomicrographs of the histopathological analysis (H E; 40x) and immunohistochemical staining showing marked tumor regression and reductions in cell proliferation (ki-67) and protein expression of L1CAM, MMP-2, MMP-9 and NF-κb in bone specimens from animals receiving L1CAM-siRNA treatment (200x; scale bar=100 μm).

    Journal: Oncotarget

    Article Title: Targeting L1 cell adhesion molecule expression using liposome-encapsulated siRNA suppresses prostate cancer bone metastasis and growth

    doi:

    Figure Lengend Snippet: Histopathologic examination of PC3 bone tumors Mice were killed on day 50 after tumor cell implantation, and tumor-bearing legs were harvested and evaluated. Representative photomicrographs of the histopathological analysis (H E; 40x) and immunohistochemical staining showing marked tumor regression and reductions in cell proliferation (ki-67) and protein expression of L1CAM, MMP-2, MMP-9 and NF-κb in bone specimens from animals receiving L1CAM-siRNA treatment (200x; scale bar=100 μm).

    Article Snippet: Gelatin zymography MMP-2 and MMP-9 enzymatic activity was determined using a 10% gelatin zymography sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (Invitrogen) according to the manufacturer's instruction.

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Expressing

    Effect of L1 cell adhesion molecule (L1CAM) shRNA on matrix metalloproteinase and nuclear factor NF-κB activation in prostate cancer PC3 cells (A) Gelatin zymographic (top) and Western blot (WB, bottom) analyses of MMP-2 and MMP-9 expression in conditioned medium (CM) from the shRNA-expressing PC3 cell lines. The positions of active MMP-2 and MMP-9 are indicated. (B) Quantitative real time RT-PCR analysis of transcriptional levels of MMP-2 and MMP-9. The relative MMP expression was normalized to the HSPCB housekeeping gene and plotted relative to the sh-NT control. Data are presented as the mean±SD of three independent determinations. ** p

    Journal: Oncotarget

    Article Title: Targeting L1 cell adhesion molecule expression using liposome-encapsulated siRNA suppresses prostate cancer bone metastasis and growth

    doi:

    Figure Lengend Snippet: Effect of L1 cell adhesion molecule (L1CAM) shRNA on matrix metalloproteinase and nuclear factor NF-κB activation in prostate cancer PC3 cells (A) Gelatin zymographic (top) and Western blot (WB, bottom) analyses of MMP-2 and MMP-9 expression in conditioned medium (CM) from the shRNA-expressing PC3 cell lines. The positions of active MMP-2 and MMP-9 are indicated. (B) Quantitative real time RT-PCR analysis of transcriptional levels of MMP-2 and MMP-9. The relative MMP expression was normalized to the HSPCB housekeeping gene and plotted relative to the sh-NT control. Data are presented as the mean±SD of three independent determinations. ** p

    Article Snippet: Gelatin zymography MMP-2 and MMP-9 enzymatic activity was determined using a 10% gelatin zymography sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (Invitrogen) according to the manufacturer's instruction.

    Techniques: shRNA, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    Highly glycosylated CD147 promotes MMPs activity in primary astrocytes. a Representative immunoblots of CD147 and caveolin-1 in AGEs and tunicamycin-treated primary astrocytes. b Densitometric quantification results of caveolin-1. c , d MMP-2/9 activity in the conditioned media. One asterisk (*) represents P

    Journal: Journal of Neuroinflammation

    Article Title: Highly glycosylated CD147 promotes hemorrhagic transformation after rt-PA treatment in diabetes: a novel therapeutic target?

    doi: 10.1186/s12974-019-1460-1

    Figure Lengend Snippet: Highly glycosylated CD147 promotes MMPs activity in primary astrocytes. a Representative immunoblots of CD147 and caveolin-1 in AGEs and tunicamycin-treated primary astrocytes. b Densitometric quantification results of caveolin-1. c , d MMP-2/9 activity in the conditioned media. One asterisk (*) represents P

    Article Snippet: Detection of MMP-2/9 activity MMP-2/9 activity was detected according to the manufacturer’s instructions (Invitrogen).

    Techniques: Activity Assay, Western Blot

    Abridged general view. Diabetes upregulated the expression of caveolin-1 and the glycosylation of CD147 in both astrocytes and endothelium and further elevated the activities of MMPs, especially MMP-2/9. Higher MMP activity accelerates the degradation of BBB components and ultimately leads to erythrocyte leakage after rt-PA treatment of acute cerebral ischemia

    Journal: Journal of Neuroinflammation

    Article Title: Highly glycosylated CD147 promotes hemorrhagic transformation after rt-PA treatment in diabetes: a novel therapeutic target?

    doi: 10.1186/s12974-019-1460-1

    Figure Lengend Snippet: Abridged general view. Diabetes upregulated the expression of caveolin-1 and the glycosylation of CD147 in both astrocytes and endothelium and further elevated the activities of MMPs, especially MMP-2/9. Higher MMP activity accelerates the degradation of BBB components and ultimately leads to erythrocyte leakage after rt-PA treatment of acute cerebral ischemia

    Article Snippet: Detection of MMP-2/9 activity MMP-2/9 activity was detected according to the manufacturer’s instructions (Invitrogen).

    Techniques: Expressing, Activity Assay

    Ex-4 ameliorates rt-PA-induced hemorrhagic transformation by downregulating HG-CD147/MMPs pathway. Ex-4 is a GLP-1R agonist that promotes insulin secretion. a , b Ex-4 treatment reduces the stroke volume and HT in both saline and rt-PA groups. c Ex-4 shows a protecting effect on BBB integrity against rt-PA in Evans blue dye assay. Data expressed as mean ± SD. d Representative immunoblots and densitometric quantifications of CD147. Asterisk denotes comparison with control group without rt-PA treatment, number sign denotes comparison with control group with rt-PA treatment, and section sign denotes comparison with Ex-4-treated group without rt-PA treatment. e , f MMP-2/9 activity. Data expressed as mean ± SEM and analyzed by two-way ANOVA. One asterisk (*) represents P

    Journal: Journal of Neuroinflammation

    Article Title: Highly glycosylated CD147 promotes hemorrhagic transformation after rt-PA treatment in diabetes: a novel therapeutic target?

    doi: 10.1186/s12974-019-1460-1

    Figure Lengend Snippet: Ex-4 ameliorates rt-PA-induced hemorrhagic transformation by downregulating HG-CD147/MMPs pathway. Ex-4 is a GLP-1R agonist that promotes insulin secretion. a , b Ex-4 treatment reduces the stroke volume and HT in both saline and rt-PA groups. c Ex-4 shows a protecting effect on BBB integrity against rt-PA in Evans blue dye assay. Data expressed as mean ± SD. d Representative immunoblots and densitometric quantifications of CD147. Asterisk denotes comparison with control group without rt-PA treatment, number sign denotes comparison with control group with rt-PA treatment, and section sign denotes comparison with Ex-4-treated group without rt-PA treatment. e , f MMP-2/9 activity. Data expressed as mean ± SEM and analyzed by two-way ANOVA. One asterisk (*) represents P

    Article Snippet: Detection of MMP-2/9 activity MMP-2/9 activity was detected according to the manufacturer’s instructions (Invitrogen).

    Techniques: Transformation Assay, Western Blot, Activity Assay

    Overexpression of N-glycosylation defective CD147 mutant N152Q blocked MMPs production in BMECs. a Representative Western blots of CD147, caveolin-1, occludin, and ZO-1. b , c Densitometric quantification results of CD147 and caveolin-1 in control and empty vector- or N152Q mutant-transfected BMECs. d , e MMP-2/9 activity in the conditioned media. f , g Densitometric quantification results of occludin and ZO-1. Data expressed as mean ± SEM and analyzed by one-way ANOVA. Asterisk (*) denotes comparison with the control group and pound (#) with the empty vector group. One asterisk (*) represents P

    Journal: Journal of Neuroinflammation

    Article Title: Highly glycosylated CD147 promotes hemorrhagic transformation after rt-PA treatment in diabetes: a novel therapeutic target?

    doi: 10.1186/s12974-019-1460-1

    Figure Lengend Snippet: Overexpression of N-glycosylation defective CD147 mutant N152Q blocked MMPs production in BMECs. a Representative Western blots of CD147, caveolin-1, occludin, and ZO-1. b , c Densitometric quantification results of CD147 and caveolin-1 in control and empty vector- or N152Q mutant-transfected BMECs. d , e MMP-2/9 activity in the conditioned media. f , g Densitometric quantification results of occludin and ZO-1. Data expressed as mean ± SEM and analyzed by one-way ANOVA. Asterisk (*) denotes comparison with the control group and pound (#) with the empty vector group. One asterisk (*) represents P

    Article Snippet: Detection of MMP-2/9 activity MMP-2/9 activity was detected according to the manufacturer’s instructions (Invitrogen).

    Techniques: Over Expression, Mutagenesis, Western Blot, Plasmid Preparation, Transfection, Activity Assay

    MMP-9 activation of endothelial cells. (A) Effect of stimulation with MMP-9 on HUVEC cell activation (pERK); n = 4. (B) Effect of stimulation with MMP-9 on HUVEC cell apoptosis (caspase-3 activity; p

    Journal: PLoS ONE

    Article Title: Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    doi: 10.1371/journal.pone.0171427

    Figure Lengend Snippet: MMP-9 activation of endothelial cells. (A) Effect of stimulation with MMP-9 on HUVEC cell activation (pERK); n = 4. (B) Effect of stimulation with MMP-9 on HUVEC cell apoptosis (caspase-3 activity; p

    Article Snippet: Western blotting HUVEC cells were incubated for 5 minutes with human enzymatically active MMP-9 at the concentration range of 0.1–0.01 nM (Calbiochem; San Diego CA).

    Techniques: Activation Assay, Activity Assay

    MMP-9 interaction with endothelial cell PAR-1. (A) Activation of PAR-1 by MMP-9 (pseudo color green); n = 3. (B) Expression of pERK in HUVEC cells by MMP-9 in presence and absence of anti-PAR-1; n = 3. (C). Expression of MMP-9 and PAR-1 in aortic leaflets from patients with atherosclerosis; n = 3.

    Journal: PLoS ONE

    Article Title: Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    doi: 10.1371/journal.pone.0171427

    Figure Lengend Snippet: MMP-9 interaction with endothelial cell PAR-1. (A) Activation of PAR-1 by MMP-9 (pseudo color green); n = 3. (B) Expression of pERK in HUVEC cells by MMP-9 in presence and absence of anti-PAR-1; n = 3. (C). Expression of MMP-9 and PAR-1 in aortic leaflets from patients with atherosclerosis; n = 3.

    Article Snippet: Western blotting HUVEC cells were incubated for 5 minutes with human enzymatically active MMP-9 at the concentration range of 0.1–0.01 nM (Calbiochem; San Diego CA).

    Techniques: Activation Assay, Expressing

    Level of MMP-9 (total and endogenous active) in plasma of WD + SHS exposed ApoE -/- mice. (A) Total MMP-9 in plasma at 7 weeks and endogenous active MMP-9 at 13 weeks exposure. Values are shown as mean ± STD, † p

    Journal: PLoS ONE

    Article Title: Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    doi: 10.1371/journal.pone.0171427

    Figure Lengend Snippet: Level of MMP-9 (total and endogenous active) in plasma of WD + SHS exposed ApoE -/- mice. (A) Total MMP-9 in plasma at 7 weeks and endogenous active MMP-9 at 13 weeks exposure. Values are shown as mean ± STD, † p

    Article Snippet: Western blotting HUVEC cells were incubated for 5 minutes with human enzymatically active MMP-9 at the concentration range of 0.1–0.01 nM (Calbiochem; San Diego CA).

    Techniques: Mouse Assay

    Mapping MMP-9 cleavage sites on PN-1. A , recombinant PN-1 (500 ng) was incubated with 100 ng of active MMP-9 at 37 °C for 20 min or 1 h. 5% of the cleaved products were resolved by 15% SDS-PAGE and immunoblotted with anti-PN-1

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: Mapping MMP-9 cleavage sites on PN-1. A , recombinant PN-1 (500 ng) was incubated with 100 ng of active MMP-9 at 37 °C for 20 min or 1 h. 5% of the cleaved products were resolved by 15% SDS-PAGE and immunoblotted with anti-PN-1

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques: Recombinant, Incubation, SDS Page

    MMP-9 knockdown and pharmacological inhibition in tumor cells: initial characterization. A , conditioned medium collected from the indicated human tumor cell lines was screened through gelatin zymography for the expression and activity of gelatinases.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: MMP-9 knockdown and pharmacological inhibition in tumor cells: initial characterization. A , conditioned medium collected from the indicated human tumor cell lines was screened through gelatin zymography for the expression and activity of gelatinases.

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques: Inhibition, Zymography, Expressing, Activity Assay

    Identification of MMP-9 Substrate Candidates by Nano-UPLC-MSE Analysis—

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: Identification of MMP-9 Substrate Candidates by Nano-UPLC-MSE Analysis—

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques:

    MMP-9-dependent degradation of PN-1 and LIF. A , recombinant PN-1 (40 ng) was incubated with 10 ng ( lanes 2 – 4 ), 2 ng ( lane 5 ), 5 ng ( lane 6 ), or 10 ng ( lane 7 ) of active MMP-9; 10 ng of pro-MMP-9 ( lane 8 ), or 10 ng of active MMP-2 ( lane 9 ) at 37

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: MMP-9-dependent degradation of PN-1 and LIF. A , recombinant PN-1 (40 ng) was incubated with 10 ng ( lanes 2 – 4 ), 2 ng ( lane 5 ), 5 ng ( lane 6 ), or 10 ng ( lane 7 ) of active MMP-9; 10 ng of pro-MMP-9 ( lane 8 ), or 10 ng of active MMP-2 ( lane 9 ) at 37

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques: Recombinant, Incubation

    Six candidate substrates of MMP-9 identified by MS were validated through immunoblot. Conditioned medium and whole cell lysates collected from PC-3ML cells treated as indicated were subjected to electrophoresis, transferred to PVDF membranes, and probed

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: Six candidate substrates of MMP-9 identified by MS were validated through immunoblot. Conditioned medium and whole cell lysates collected from PC-3ML cells treated as indicated were subjected to electrophoresis, transferred to PVDF membranes, and probed

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques: Mass Spectrometry, Electrophoresis

    Cleavage topology of collagen VI by MMP-9. A , collagen VI sequence coverage obtained by nano-UPLC-MS E analysis of trypsin-digested cell culture supernatants from control ( underlined ) and MMP-9 KD samples ( black letters ). B , relative quantitation of individual

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: Cleavage topology of collagen VI by MMP-9. A , collagen VI sequence coverage obtained by nano-UPLC-MS E analysis of trypsin-digested cell culture supernatants from control ( underlined ) and MMP-9 KD samples ( black letters ). B , relative quantitation of individual

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques: Sequencing, Mass Spectrometry, Cell Culture, Quantitation Assay

    A label-free quantitative proteomics approach for MMP-9 substrate degradomics. Cell culture supernatants of PC3-ML control and those treated either with BB-94 inhibitor or induction of MMP-9 RNA interference ( RNAi ) knockdown were harvested and subjected

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach *Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach * S⃞

    doi: 10.1074/mcp.M800095-MCP200

    Figure Lengend Snippet: A label-free quantitative proteomics approach for MMP-9 substrate degradomics. Cell culture supernatants of PC3-ML control and those treated either with BB-94 inhibitor or induction of MMP-9 RNA interference ( RNAi ) knockdown were harvested and subjected

    Article Snippet: Human active MMP-9, MMP-2, or pro-MMP-9 (Calbiochem) was tested for the ability to cleave human recombinant LIF (Chemicon) or PN-1 protein (R & D Systems) in vitro .

    Techniques: Cell Culture

    Endogenous ERK1/2 and MMP-9 in axotomized rat sciatic nerve

    Journal: Glia

    Article Title: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway

    doi: 10.1002/glia.20851

    Figure Lengend Snippet: Endogenous ERK1/2 and MMP-9 in axotomized rat sciatic nerve

    Article Snippet: Bovine serum albumin (BSA, 100 μg/ml, Sigma), recombinant rat tumor necrosis factor alpha (TNF-α, 10 ng/ml, R & D), lipopolysaccharide (LPS, 100 ng/ml, Sigma), recombinant murine 7S nerve growth factor (NGF, 100 ng/ml, Invitrogen), recombinant human neuregulin 1 (NRG-1, 10 ng/ml, R & D Systems) and recombinant human active MMP-9 (rhMMP-9, Calbiochem).

    Techniques:

    MMP-9 activation of trophic signaling in Schwann cells (a schematic diagram)

    Journal: Glia

    Article Title: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway

    doi: 10.1002/glia.20851

    Figure Lengend Snippet: MMP-9 activation of trophic signaling in Schwann cells (a schematic diagram)

    Article Snippet: Bovine serum albumin (BSA, 100 μg/ml, Sigma), recombinant rat tumor necrosis factor alpha (TNF-α, 10 ng/ml, R & D), lipopolysaccharide (LPS, 100 ng/ml, Sigma), recombinant murine 7S nerve growth factor (NGF, 100 ng/ml, Invitrogen), recombinant human neuregulin 1 (NRG-1, 10 ng/ml, R & D Systems) and recombinant human active MMP-9 (rhMMP-9, Calbiochem).

    Techniques: Activation Assay

    MMP-9 inhibits Schwann cell proliferation in vitro

    Journal: Glia

    Article Title: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway

    doi: 10.1002/glia.20851

    Figure Lengend Snippet: MMP-9 inhibits Schwann cell proliferation in vitro

    Article Snippet: Bovine serum albumin (BSA, 100 μg/ml, Sigma), recombinant rat tumor necrosis factor alpha (TNF-α, 10 ng/ml, R & D), lipopolysaccharide (LPS, 100 ng/ml, Sigma), recombinant murine 7S nerve growth factor (NGF, 100 ng/ml, Invitrogen), recombinant human neuregulin 1 (NRG-1, 10 ng/ml, R & D Systems) and recombinant human active MMP-9 (rhMMP-9, Calbiochem).

    Techniques: In Vitro

    Schwann cell proliferation in axotomized nerves of MMP-9 −/− knockout mice

    Journal: Glia

    Article Title: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway

    doi: 10.1002/glia.20851

    Figure Lengend Snippet: Schwann cell proliferation in axotomized nerves of MMP-9 −/− knockout mice

    Article Snippet: Bovine serum albumin (BSA, 100 μg/ml, Sigma), recombinant rat tumor necrosis factor alpha (TNF-α, 10 ng/ml, R & D), lipopolysaccharide (LPS, 100 ng/ml, Sigma), recombinant murine 7S nerve growth factor (NGF, 100 ng/ml, Invitrogen), recombinant human neuregulin 1 (NRG-1, 10 ng/ml, R & D Systems) and recombinant human active MMP-9 (rhMMP-9, Calbiochem).

    Techniques: Knock-Out, Mouse Assay

    MMP-9 activates ERK1/2 and suppresses Schwann cell growth

    Journal: Glia

    Article Title: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway

    doi: 10.1002/glia.20851

    Figure Lengend Snippet: MMP-9 activates ERK1/2 and suppresses Schwann cell growth

    Article Snippet: Bovine serum albumin (BSA, 100 μg/ml, Sigma), recombinant rat tumor necrosis factor alpha (TNF-α, 10 ng/ml, R & D), lipopolysaccharide (LPS, 100 ng/ml, Sigma), recombinant murine 7S nerve growth factor (NGF, 100 ng/ml, Invitrogen), recombinant human neuregulin 1 (NRG-1, 10 ng/ml, R & D Systems) and recombinant human active MMP-9 (rhMMP-9, Calbiochem).

    Techniques:

    MMP-9 expression in primary Schwann cells

    Journal: Glia

    Article Title: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway

    doi: 10.1002/glia.20851

    Figure Lengend Snippet: MMP-9 expression in primary Schwann cells

    Article Snippet: Bovine serum albumin (BSA, 100 μg/ml, Sigma), recombinant rat tumor necrosis factor alpha (TNF-α, 10 ng/ml, R & D), lipopolysaccharide (LPS, 100 ng/ml, Sigma), recombinant murine 7S nerve growth factor (NGF, 100 ng/ml, Invitrogen), recombinant human neuregulin 1 (NRG-1, 10 ng/ml, R & D Systems) and recombinant human active MMP-9 (rhMMP-9, Calbiochem).

    Techniques: Expressing

    Expression of TIMP-1 and MMPs in primary cortical neurons. A. Semi-quantitative RT-PCR followed by separation of the PCR products in ethidium bromide stained gels showing expression by cortical neurons 24 h after seeding and by N2a neuroblastoma cells of mRNAs encoding endogenous TIMP-1, MMP-2, MMP-9 and MT1-MMP after 35 cycles of PCR. Note that in cortical neurons, MMP-9 mRNA is barely detected as compared with N2a cells and with the other neuronal mRNAs which were already detected at 30 cycles of PCR. GAPDH (30 cycles) was used as a standard; 100 bp molecular weight ladder (M). B. Western blot showing TIMP-1 immunoreactivity after equal protein loading from supernatants of cortical neurons at different times after adding 2.5 nM of mrTIMP-1, or from control untreated cultures at the same time points, 0, 24 and 72 h. Note that endogenous TIMP-1 is not detected in untreated cultures, whereas mrTIMP-1 is easily detected in the supernatants of these cultures even after 72 h, indicating high stability of the protein. C. Fluorescent microphotographs of cortical neurons showing βIII-tubulin (green) and TIMP-1 (red) immunostaining co-labelled with the nuclear marker Hoechst (blue) 24 h after seeding. Note low levels of endogenous TIMP-1, slightly above background levels (Blank) only in the soma of some cells. Scale bar 20 µm. Figures are representative of at least 3 independent experiments.

    Journal: PLoS ONE

    Article Title: A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

    doi: 10.1371/journal.pone.0008289

    Figure Lengend Snippet: Expression of TIMP-1 and MMPs in primary cortical neurons. A. Semi-quantitative RT-PCR followed by separation of the PCR products in ethidium bromide stained gels showing expression by cortical neurons 24 h after seeding and by N2a neuroblastoma cells of mRNAs encoding endogenous TIMP-1, MMP-2, MMP-9 and MT1-MMP after 35 cycles of PCR. Note that in cortical neurons, MMP-9 mRNA is barely detected as compared with N2a cells and with the other neuronal mRNAs which were already detected at 30 cycles of PCR. GAPDH (30 cycles) was used as a standard; 100 bp molecular weight ladder (M). B. Western blot showing TIMP-1 immunoreactivity after equal protein loading from supernatants of cortical neurons at different times after adding 2.5 nM of mrTIMP-1, or from control untreated cultures at the same time points, 0, 24 and 72 h. Note that endogenous TIMP-1 is not detected in untreated cultures, whereas mrTIMP-1 is easily detected in the supernatants of these cultures even after 72 h, indicating high stability of the protein. C. Fluorescent microphotographs of cortical neurons showing βIII-tubulin (green) and TIMP-1 (red) immunostaining co-labelled with the nuclear marker Hoechst (blue) 24 h after seeding. Note low levels of endogenous TIMP-1, slightly above background levels (Blank) only in the soma of some cells. Scale bar 20 µm. Figures are representative of at least 3 independent experiments.

    Article Snippet: Equal amounts of protein were subjected to 8.5% SDS-PAGE containing 2.25 mg/ml porcine gelatin and 160 ng/ml of human recombinant active MMP-9 (Chemicon).

    Techniques: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Staining, Molecular Weight, Western Blot, Immunostaining, Marker

    Effects of TIMP-1/GFP fusion protein on the morphology and neurite development of cortical neurons. A. Reverse gel zymography from supernatants of N2a cells transfected with TIMP-1/GFP fusion protein demonstrating that TIMP-1/GFP retains inhibitory activity on MMP proteolytic activity. Note that the band of inhibited MMP-9 activity corresponds to the size of TIMP-1 (33 kDa) + GFP (28 kDa). B. Examples of cortical neurons transfected with GFP or TIMP-1/GFP and immunostained with an anti-GFP antibody 48 h after transfection. White fluorescence photomicrographs were converted to black negative images to improve the details on the neuritic arbour. Note that TIMP-1 overexpression in these neurons strongly reduces the complexity of their arborisation. Scale bar 20 µm. C and D. Quantification of the changes induced by TIMP-1/GFP overexpression compared to GFP alone. The values represent the means ± SEM of 4 independent experiments. *p

    Journal: PLoS ONE

    Article Title: A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

    doi: 10.1371/journal.pone.0008289

    Figure Lengend Snippet: Effects of TIMP-1/GFP fusion protein on the morphology and neurite development of cortical neurons. A. Reverse gel zymography from supernatants of N2a cells transfected with TIMP-1/GFP fusion protein demonstrating that TIMP-1/GFP retains inhibitory activity on MMP proteolytic activity. Note that the band of inhibited MMP-9 activity corresponds to the size of TIMP-1 (33 kDa) + GFP (28 kDa). B. Examples of cortical neurons transfected with GFP or TIMP-1/GFP and immunostained with an anti-GFP antibody 48 h after transfection. White fluorescence photomicrographs were converted to black negative images to improve the details on the neuritic arbour. Note that TIMP-1 overexpression in these neurons strongly reduces the complexity of their arborisation. Scale bar 20 µm. C and D. Quantification of the changes induced by TIMP-1/GFP overexpression compared to GFP alone. The values represent the means ± SEM of 4 independent experiments. *p

    Article Snippet: Equal amounts of protein were subjected to 8.5% SDS-PAGE containing 2.25 mg/ml porcine gelatin and 160 ng/ml of human recombinant active MMP-9 (Chemicon).

    Techniques: Zymography, Transfection, Activity Assay, Fluorescence, Over Expression

    Expression of MMP-2 and MMP-9 in cortical neurons 24 h after seeding. A. Gelatin zymograms of MMP-2 and MMP-9 from supernatant of cortical neurons, cytosol and membrane fractions. Human active recombinant MMP-2 (hrMMP-2) was used as a positive control (200 pg). The zymogram shows much higher expression of MMP-2 (∼70 kDa) than MMP-9 (∼100 kDa) in the supernatant, higher expression of MMP-2 in the cytosol where MMP-9 is barely detectable, and rather similar intensity of gelatinolytic bands for both gelatinases in the membrane fraction. B. Quantification of the MMP-2/MMP-9 ratio from zymograms, where active recombinant MMP-2 was used as normalising control. Values represent the means ± SEM of 3 independent experiments. Note that the quantification takes into consideration a 5-fold higher gelatinolytic activity of MMP-9 than MMP-2. C. Western blot demonstrating MMP-2 immunoreactivity in the supernatants of cortical neurons after enrichment and precipitation of the samples with gelatin beads. Active human recombinant MMP-2 (5 ng) was used as a positive control. Images are representative of 3 independent experiments. D. Fluorescence microphotographs showing immunolabelling of MMP-2 (green) and phalloidin F-actin labelling (red) in cortical neurons 24 h after seeding. Hoechst #33258 stained the nuclei (blue). Note that MMP-2 is distributed in the cell body and neurites. In the growth cones, MMP-2 is mainly located to the central domain (insets with high power magnifications in the lower row) and virtually excluded from F-actin rich areas, notably the peripheral domain. Scale bars are 20 µm for entire neurons and 10 µm for close up of growth cones.

    Journal: PLoS ONE

    Article Title: A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

    doi: 10.1371/journal.pone.0008289

    Figure Lengend Snippet: Expression of MMP-2 and MMP-9 in cortical neurons 24 h after seeding. A. Gelatin zymograms of MMP-2 and MMP-9 from supernatant of cortical neurons, cytosol and membrane fractions. Human active recombinant MMP-2 (hrMMP-2) was used as a positive control (200 pg). The zymogram shows much higher expression of MMP-2 (∼70 kDa) than MMP-9 (∼100 kDa) in the supernatant, higher expression of MMP-2 in the cytosol where MMP-9 is barely detectable, and rather similar intensity of gelatinolytic bands for both gelatinases in the membrane fraction. B. Quantification of the MMP-2/MMP-9 ratio from zymograms, where active recombinant MMP-2 was used as normalising control. Values represent the means ± SEM of 3 independent experiments. Note that the quantification takes into consideration a 5-fold higher gelatinolytic activity of MMP-9 than MMP-2. C. Western blot demonstrating MMP-2 immunoreactivity in the supernatants of cortical neurons after enrichment and precipitation of the samples with gelatin beads. Active human recombinant MMP-2 (5 ng) was used as a positive control. Images are representative of 3 independent experiments. D. Fluorescence microphotographs showing immunolabelling of MMP-2 (green) and phalloidin F-actin labelling (red) in cortical neurons 24 h after seeding. Hoechst #33258 stained the nuclei (blue). Note that MMP-2 is distributed in the cell body and neurites. In the growth cones, MMP-2 is mainly located to the central domain (insets with high power magnifications in the lower row) and virtually excluded from F-actin rich areas, notably the peripheral domain. Scale bars are 20 µm for entire neurons and 10 µm for close up of growth cones.

    Article Snippet: Equal amounts of protein were subjected to 8.5% SDS-PAGE containing 2.25 mg/ml porcine gelatin and 160 ng/ml of human recombinant active MMP-9 (Chemicon).

    Techniques: Expressing, Recombinant, Positive Control, Activity Assay, Western Blot, Fluorescence, Staining

    Enzyme specificity in inhibitory activity of APP-IP-TIMP-2. A , MMP-1 (0.8 n m ), MMP-2 (0.2 n m ), MMP-3 (27 n m ), MMP-7 (1.5 n m ), MMP-8 (7.2 n m ), MMP-9 (1.4 n m ), and the catalytic domain of MT1-MMP (MT1cat, 1.0 n m ) were incubated with 50 μ m 3163v,

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Design of a Highly Selective and Strong Protein Inhibitor against Matrix Metalloproteinase-2 (MMP-2) *

    doi: 10.1074/jbc.M112.441758

    Figure Lengend Snippet: Enzyme specificity in inhibitory activity of APP-IP-TIMP-2. A , MMP-1 (0.8 n m ), MMP-2 (0.2 n m ), MMP-3 (27 n m ), MMP-7 (1.5 n m ), MMP-8 (7.2 n m ), MMP-9 (1.4 n m ), and the catalytic domain of MT1-MMP (MT1cat, 1.0 n m ) were incubated with 50 μ m 3163v,

    Article Snippet: The sources of materials used are as follows: pcDNA3.1/Zeo (−) and Lipofectamine LTX Reagent from Invitrogen (Carlsbad, CA); pGEM3z from Promega (Madison, WI); gelatin-Sepharose 4B, heparin-Sepharose CL-6B, and CNBr-activated Sepharose 4B from GE Healthcare UK Ltd. (Amersham Biosciences); the synthetic substrate for MMPs, 3163v (7-methoxycoumarin-4-yl)-acetyl-Pro-Leu-Gly-Leu-[ N β -(2,4-dinitrophenyl)- l -2,3-diaminopropionyl]-Ala-Arg amide), 3226v (7-methoxycoumarin-4-yl)-acetyl-Lys-Pro-Leu-Gly-Leu-[ N β -(2,4-dinitrophenyl)- l -2,3-diaminopropionyl]-Ala-Arg amide) and the synthetic MMPs inhibitor TAPI-1 ( N -( R )-(2-(hydroxaminocarbonyl)methyl)-4-methylpentanoyl- l -naphthylalanyl- l -alanine-2-aminoethyl amide) from Peptide Institute, Inc. (Osaka, Japan); p -aminophenyl mercuric acetate (APMA) from Tokyo Kasei (Tokyo, Japan); purified human pro-MMP-1, human pro-MMP-8, and human ADAM17 from Calbiochem (La Jolla, CA); purified human pro-MMP-3, human pro-MMP-9, and the active catalytic domain of human MT1-MMP from EMD Millipore Co. (Billerica, MA); the plant lectin concanavalin A (Con A, type IV, substantially free of carbohydrates) was from Sigma; gelatin from Difco (Detroit, MI); bovine type IV collagen from Nitta gelatin (Osaka).

    Techniques: Activity Assay, Incubation

    OGD-induced occludin degradation is MMP-2/9 dependent. A) OGD rapidly elevated MMP-2/9 levels in conditioned media. After exposure of bEND3 cells to OGD for 2 h, a significant increase in MMP-2/9 levels was detected in the conditioned medium on gelatin

    Journal: The Journal of Neuroscience

    Article Title: Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage

    doi: 10.1523/JNEUROSCI.6409-11.2012

    Figure Lengend Snippet: OGD-induced occludin degradation is MMP-2/9 dependent. A) OGD rapidly elevated MMP-2/9 levels in conditioned media. After exposure of bEND3 cells to OGD for 2 h, a significant increase in MMP-2/9 levels was detected in the conditioned medium on gelatin

    Article Snippet: A mixture of human MMP-2/9 (Chemicon) was used as gelatinase standards.

    Techniques:

    OGD elevates extracellular MMP-2/9 levels through promoting their secretion from the pre-existing intracellular pool in bEND3 cells. A) Gelatin zymography analysis showed that 2-h OGD markedly increased MMP-2/9 levels in the conditioned medium (CM), which

    Journal: The Journal of Neuroscience

    Article Title: Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage

    doi: 10.1523/JNEUROSCI.6409-11.2012

    Figure Lengend Snippet: OGD elevates extracellular MMP-2/9 levels through promoting their secretion from the pre-existing intracellular pool in bEND3 cells. A) Gelatin zymography analysis showed that 2-h OGD markedly increased MMP-2/9 levels in the conditioned medium (CM), which

    Article Snippet: A mixture of human MMP-2/9 (Chemicon) was used as gelatinase standards.

    Techniques: Zymography

    Inhibition of MMP-2/9 with SB-3CT or knockdown of Cav-1 with siRNA reduces OGD-induced BBB disruption in vitro . The permeability of FITC-dextran across bEND3 monolayers was significantly increased after 2-h exposure to OGD, which was partially inhibited

    Journal: The Journal of Neuroscience

    Article Title: Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage

    doi: 10.1523/JNEUROSCI.6409-11.2012

    Figure Lengend Snippet: Inhibition of MMP-2/9 with SB-3CT or knockdown of Cav-1 with siRNA reduces OGD-induced BBB disruption in vitro . The permeability of FITC-dextran across bEND3 monolayers was significantly increased after 2-h exposure to OGD, which was partially inhibited

    Article Snippet: A mixture of human MMP-2/9 (Chemicon) was used as gelatinase standards.

    Techniques: Inhibition, In Vitro, Permeability

    Cerebral ischemia rapidly increases extracellular MMP-2/9 levels and their activities in ischemic striatum. After 2 h-MCAO, the gelatinolytic activity of MMP-2/9 and their extracellular levels were analyzed by in situ zymography and microdialysis sampling/gel

    Journal: The Journal of Neuroscience

    Article Title: Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage

    doi: 10.1523/JNEUROSCI.6409-11.2012

    Figure Lengend Snippet: Cerebral ischemia rapidly increases extracellular MMP-2/9 levels and their activities in ischemic striatum. After 2 h-MCAO, the gelatinolytic activity of MMP-2/9 and their extracellular levels were analyzed by in situ zymography and microdialysis sampling/gel

    Article Snippet: A mixture of human MMP-2/9 (Chemicon) was used as gelatinase standards.

    Techniques: Activity Assay, In Situ, Zymography, Sampling

    Expression of the pro- and active forms of MMP-9 protein in the rat small bowel muscularis 14 hr after surgical manipulation. MMP-9 expression was assessed in tissue lysates by Western blot analysis. A. Tissue lysates probed with mAb L51/82 recognizing

    Journal: Gastroenterology

    Article Title: Matrix Metalloproteinase-9 Inhibition Reduces Inflammation and Improves Motility in Murine Models of Post-Operative Ileus

    doi: 10.1053/j.gastro.2011.06.035

    Figure Lengend Snippet: Expression of the pro- and active forms of MMP-9 protein in the rat small bowel muscularis 14 hr after surgical manipulation. MMP-9 expression was assessed in tissue lysates by Western blot analysis. A. Tissue lysates probed with mAb L51/82 recognizing

    Article Snippet: Rat small bowel muscularis whole mounts were harvested 14 hr postoperatively and pretreated for 30 min with anti-human MMP-9 (Calbiochem, clone 6-6B) or MMP-2 (Millipore, clone CA-4001) active site neutralizing antibodies, or PBS.

    Techniques: Expressing, Western Blot

    MMP-9 KO mice are resistant to postoperative ileus (POI). A. Representative digital photomicrographs of small bowel muscularis whole mount harvested 24 hr postoperatively. Myeloperoxidase (MPO) positive leukocytes were absent in non-operated control animals,

    Journal: Gastroenterology

    Article Title: Matrix Metalloproteinase-9 Inhibition Reduces Inflammation and Improves Motility in Murine Models of Post-Operative Ileus

    doi: 10.1053/j.gastro.2011.06.035

    Figure Lengend Snippet: MMP-9 KO mice are resistant to postoperative ileus (POI). A. Representative digital photomicrographs of small bowel muscularis whole mount harvested 24 hr postoperatively. Myeloperoxidase (MPO) positive leukocytes were absent in non-operated control animals,

    Article Snippet: Rat small bowel muscularis whole mounts were harvested 14 hr postoperatively and pretreated for 30 min with anti-human MMP-9 (Calbiochem, clone 6-6B) or MMP-2 (Millipore, clone CA-4001) active site neutralizing antibodies, or PBS.

    Techniques: Mouse Assay

    Real time RT-PCR analysis of the effects of iNOS gene deletion on peak MMP-9 and TIMP-1 gene expression (12 hr postoperatively). A. Time course analysis of iNOS gene expression following surgical manipulation in the wild type mouse shows significant induction

    Journal: Gastroenterology

    Article Title: Matrix Metalloproteinase-9 Inhibition Reduces Inflammation and Improves Motility in Murine Models of Post-Operative Ileus

    doi: 10.1053/j.gastro.2011.06.035

    Figure Lengend Snippet: Real time RT-PCR analysis of the effects of iNOS gene deletion on peak MMP-9 and TIMP-1 gene expression (12 hr postoperatively). A. Time course analysis of iNOS gene expression following surgical manipulation in the wild type mouse shows significant induction

    Article Snippet: Rat small bowel muscularis whole mounts were harvested 14 hr postoperatively and pretreated for 30 min with anti-human MMP-9 (Calbiochem, clone 6-6B) or MMP-2 (Millipore, clone CA-4001) active site neutralizing antibodies, or PBS.

    Techniques: Quantitative RT-PCR, Expressing

    Inhibition of MMP-9 catalytic activity protects wild type mice from intestinal smooth muscle dysmotility. A. Treatment with MMP-2/MMP-9 II (MMPi; 10 mg/kg) inhibited the influx of myeloperoxidase (MPO)-positive leukocytes into the small bowel and colonic

    Journal: Gastroenterology

    Article Title: Matrix Metalloproteinase-9 Inhibition Reduces Inflammation and Improves Motility in Murine Models of Post-Operative Ileus

    doi: 10.1053/j.gastro.2011.06.035

    Figure Lengend Snippet: Inhibition of MMP-9 catalytic activity protects wild type mice from intestinal smooth muscle dysmotility. A. Treatment with MMP-2/MMP-9 II (MMPi; 10 mg/kg) inhibited the influx of myeloperoxidase (MPO)-positive leukocytes into the small bowel and colonic

    Article Snippet: Rat small bowel muscularis whole mounts were harvested 14 hr postoperatively and pretreated for 30 min with anti-human MMP-9 (Calbiochem, clone 6-6B) or MMP-2 (Millipore, clone CA-4001) active site neutralizing antibodies, or PBS.

    Techniques: Inhibition, Activity Assay, Mouse Assay

    Real time RT-PCR time course analysis of gene expression in the mouse small bowel and colonic muscularis 3, 6, 12, and 24 hr after surgical manipulation. The induction of MMP-9 and TIMP-1 was time-dependent; reaching peak levels 12-24 hr postoperatively.

    Journal: Gastroenterology

    Article Title: Matrix Metalloproteinase-9 Inhibition Reduces Inflammation and Improves Motility in Murine Models of Post-Operative Ileus

    doi: 10.1053/j.gastro.2011.06.035

    Figure Lengend Snippet: Real time RT-PCR time course analysis of gene expression in the mouse small bowel and colonic muscularis 3, 6, 12, and 24 hr after surgical manipulation. The induction of MMP-9 and TIMP-1 was time-dependent; reaching peak levels 12-24 hr postoperatively.

    Article Snippet: Rat small bowel muscularis whole mounts were harvested 14 hr postoperatively and pretreated for 30 min with anti-human MMP-9 (Calbiochem, clone 6-6B) or MMP-2 (Millipore, clone CA-4001) active site neutralizing antibodies, or PBS.

    Techniques: Quantitative RT-PCR, Expressing

    The effect of diabetes on NGAL and MMP-9 expression in PVC implant cells and wound fluids at days 3, 6 and 12. (A) NGAL, MMP-9 and MMP-8 mRNA by qRT-PCR, (B) A representative zymogram of wound fluid at day 6, (C-E) Group data for zymography results expressed as Area Under Curve (AUC; determined from band intensity) (C) total MMP-9 (TMMP-9 = proMMP-9 +active MMP-9), (D) active MMP-9, (E) proMMP-9 and (F) NGAL/MMP-9 complex according to molecular weight. Results are from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals and are expressed as Mean ± SEM *P

    Journal: PLoS ONE

    Article Title: Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats

    doi: 10.1371/journal.pone.0170951

    Figure Lengend Snippet: The effect of diabetes on NGAL and MMP-9 expression in PVC implant cells and wound fluids at days 3, 6 and 12. (A) NGAL, MMP-9 and MMP-8 mRNA by qRT-PCR, (B) A representative zymogram of wound fluid at day 6, (C-E) Group data for zymography results expressed as Area Under Curve (AUC; determined from band intensity) (C) total MMP-9 (TMMP-9 = proMMP-9 +active MMP-9), (D) active MMP-9, (E) proMMP-9 and (F) NGAL/MMP-9 complex according to molecular weight. Results are from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals and are expressed as Mean ± SEM *P

    Article Snippet: Bands corresponding to NGAL/MMP-9 complex (~115,125 kDa), pro-MMP-9 (~92 kDa) and active-MMP-9 (~82 kDa) were observed and their identity was confirmed by Western blot analysis using antiNGAL (ab63929, Abcam) and antiMMP-9 antibodies (ab38898, Abcam) (data not shown).

    Techniques: Expressing, Quantitative RT-PCR, Zymography, Molecular Weight

    Peripheral blood neutrophil NGAL and MMP-9 at days 3, 6 and 12 post-implant surgery. (A) Neutrophil number as measured by flow cytometry and (B) Gene expression data for NGAL, MMP-9 and MMP-8. (C) Representative and group data for neutrophil NGAL and MMP-9 and their co-localisation at day 6. Results from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals are expressed as Mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats

    doi: 10.1371/journal.pone.0170951

    Figure Lengend Snippet: Peripheral blood neutrophil NGAL and MMP-9 at days 3, 6 and 12 post-implant surgery. (A) Neutrophil number as measured by flow cytometry and (B) Gene expression data for NGAL, MMP-9 and MMP-8. (C) Representative and group data for neutrophil NGAL and MMP-9 and their co-localisation at day 6. Results from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals are expressed as Mean ± SEM. * P

    Article Snippet: Bands corresponding to NGAL/MMP-9 complex (~115,125 kDa), pro-MMP-9 (~92 kDa) and active-MMP-9 (~82 kDa) were observed and their identity was confirmed by Western blot analysis using antiNGAL (ab63929, Abcam) and antiMMP-9 antibodies (ab38898, Abcam) (data not shown).

    Techniques: Flow Cytometry, Cytometry, Expressing

    The effect of diabetes on NGAL and MMP-9 in skin wound tissue at day 6. (A) Representative images for skin wound tissue NGAL and MMP-9 respectively (B) Group data for wound NGAL, and MMP-9 mRNA and (C) Group data for percentage MMP-9 staining cells in the granulation tissue. Results are from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals and are expressed as Mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats

    doi: 10.1371/journal.pone.0170951

    Figure Lengend Snippet: The effect of diabetes on NGAL and MMP-9 in skin wound tissue at day 6. (A) Representative images for skin wound tissue NGAL and MMP-9 respectively (B) Group data for wound NGAL, and MMP-9 mRNA and (C) Group data for percentage MMP-9 staining cells in the granulation tissue. Results are from Control (CON) diabetic (DM) and Insulin treated DM (DM+INS) animals and are expressed as Mean ± SEM. * P

    Article Snippet: Bands corresponding to NGAL/MMP-9 complex (~115,125 kDa), pro-MMP-9 (~92 kDa) and active-MMP-9 (~82 kDa) were observed and their identity was confirmed by Western blot analysis using antiNGAL (ab63929, Abcam) and antiMMP-9 antibodies (ab38898, Abcam) (data not shown).

    Techniques: Staining

    The effect of incubation in high glucose concentration (25mM) on neutrophil (A) NGAL, MMP-9 and MMP-8 mRNA and (B) TLR4, TLR2 and TNFα mRNA. Results are expressed as Mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats

    doi: 10.1371/journal.pone.0170951

    Figure Lengend Snippet: The effect of incubation in high glucose concentration (25mM) on neutrophil (A) NGAL, MMP-9 and MMP-8 mRNA and (B) TLR4, TLR2 and TNFα mRNA. Results are expressed as Mean ± SEM. * P

    Article Snippet: Bands corresponding to NGAL/MMP-9 complex (~115,125 kDa), pro-MMP-9 (~92 kDa) and active-MMP-9 (~82 kDa) were observed and their identity was confirmed by Western blot analysis using antiNGAL (ab63929, Abcam) and antiMMP-9 antibodies (ab38898, Abcam) (data not shown).

    Techniques: Incubation, Concentration Assay

    The effect of diabetes on PVC implant cell expression of MPO, NGAL and MMP-9 and the co-localisation of MPO with NGAL or MMP-9. (A) Representative day 6 images of implant cells stained with MPO and NGAL either alone or in combination. Group data for percentage of implant cells expressing (B) MPO, NGAL, or MMP-9 or (C) MPO in combination with either NGAL or MMP-9. Data are from the cellular fraction of implants obtained from Control (CON) Diabetic (DM) and Insulin treated DM (DM+INS) animals at days 3, 6 and 12 post implantation. Results are expressed as Mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats

    doi: 10.1371/journal.pone.0170951

    Figure Lengend Snippet: The effect of diabetes on PVC implant cell expression of MPO, NGAL and MMP-9 and the co-localisation of MPO with NGAL or MMP-9. (A) Representative day 6 images of implant cells stained with MPO and NGAL either alone or in combination. Group data for percentage of implant cells expressing (B) MPO, NGAL, or MMP-9 or (C) MPO in combination with either NGAL or MMP-9. Data are from the cellular fraction of implants obtained from Control (CON) Diabetic (DM) and Insulin treated DM (DM+INS) animals at days 3, 6 and 12 post implantation. Results are expressed as Mean ± SEM. * P

    Article Snippet: Bands corresponding to NGAL/MMP-9 complex (~115,125 kDa), pro-MMP-9 (~92 kDa) and active-MMP-9 (~82 kDa) were observed and their identity was confirmed by Western blot analysis using antiNGAL (ab63929, Abcam) and antiMMP-9 antibodies (ab38898, Abcam) (data not shown).

    Techniques: Expressing, Staining

    GS-5745 prevents activation of pro-MMP9 by MMP3. A , activation of pro-MMP9 by MMP3 was measured by incubating a fixed concentration pro-MMP9 (2.5 ng/μl MMP9-pro-cat) with MMP3 (0.25 ng/μl) in the presence of GS-5745 (30 ng/μl) or control IgG4 (30 ng/μl). Reactions were incubated at 37 °C for the indicated time, and pro- and active MMP9 were visualized by immunoblotting analysis using an anti-MMP9 antibody. B , the relative intensity of pro-MMP9 in the presence of control IgG4 (○) or GS-5745 (●) in A , normalized to the control lane (0 h), was quantified by densitometry. C , the relative intensity of active MMP9-G100L in the presence of control IgG4 (□) or GS-5745 (■) in A , normalized to the control lane (0 h), was quantified by densitometry. Each data point represents the average of duplicate experiments, and error bars represent S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9

    doi: 10.1074/jbc.M116.760579

    Figure Lengend Snippet: GS-5745 prevents activation of pro-MMP9 by MMP3. A , activation of pro-MMP9 by MMP3 was measured by incubating a fixed concentration pro-MMP9 (2.5 ng/μl MMP9-pro-cat) with MMP3 (0.25 ng/μl) in the presence of GS-5745 (30 ng/μl) or control IgG4 (30 ng/μl). Reactions were incubated at 37 °C for the indicated time, and pro- and active MMP9 were visualized by immunoblotting analysis using an anti-MMP9 antibody. B , the relative intensity of pro-MMP9 in the presence of control IgG4 (○) or GS-5745 (●) in A , normalized to the control lane (0 h), was quantified by densitometry. C , the relative intensity of active MMP9-G100L in the presence of control IgG4 (□) or GS-5745 (■) in A , normalized to the control lane (0 h), was quantified by densitometry. Each data point represents the average of duplicate experiments, and error bars represent S.D.

    Article Snippet: Reactions were incubated at 37 °C for various times, and pro- and active MMP9 were visualized by immunoblotting analysis using a rabbit monoclonal antibody raised against MMP9 (Abcam).

    Techniques: Activation Assay, Concentration Assay, Incubation

    Structure of GS-5745·MMP9-pro-cat and MMP9-pro-cat. A , the GS-5745·MMP9-pro-cat complex is represented as a ribbon diagram. The prodomain ( pro ) of MMP9 is colored yellow , and the catalytic domain ( cat ) is colored green . The heavy chain ( Hc ) of GS-5745 is colored pink , and the light chain ( Lc ) is colored cyan . Calcium atoms are colored gray , and zinc atoms are colored magenta. B , superposition of apo MMP9-pro-cat ( gray ) with MMP9-pro-cat bound to GS-5745 ( yellow and green ).

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9

    doi: 10.1074/jbc.M116.760579

    Figure Lengend Snippet: Structure of GS-5745·MMP9-pro-cat and MMP9-pro-cat. A , the GS-5745·MMP9-pro-cat complex is represented as a ribbon diagram. The prodomain ( pro ) of MMP9 is colored yellow , and the catalytic domain ( cat ) is colored green . The heavy chain ( Hc ) of GS-5745 is colored pink , and the light chain ( Lc ) is colored cyan . Calcium atoms are colored gray , and zinc atoms are colored magenta. B , superposition of apo MMP9-pro-cat ( gray ) with MMP9-pro-cat bound to GS-5745 ( yellow and green ).

    Article Snippet: Reactions were incubated at 37 °C for various times, and pro- and active MMP9 were visualized by immunoblotting analysis using a rabbit monoclonal antibody raised against MMP9 (Abcam).

    Techniques:

    GS-5745 prevents activation of pro-MMP9 by ulcerative colitis lysates. A , colon tissue was lysed, and MMP9 was visualized by immunoblotting. The upper band (molecular mass, ∼92 kDa) was detected in diseased and healthy colon lysate, whereas the lower band (∼82 kDa) was detected in only the ulcerative colitis lysate. B , endogenous MMP9 activity from each colon lysate was assessed. A standard curve of quantitatively activated MMP9 was used to determine the equivalents of active MMP9 in ng/ml. C , incubation of recombinant pro-MMP9 with ulcerative colitis lysates results in MMP9 activation. The activated MMP9 was detected by the neoepitope antibody ( red ), whereas both pro- and active MMP9 are detected by the total MMP9 antibody ( green ). MMP9 activation is inhibited by GS-5745 but not a human IgG4 isotype control antibody. D , the activity of MMP9 in C was measured with the Biotrak MMP9 assay kit. GS-5745, but not the isotype control IgG4, blocks this proteolysis. MMP9 was isolated from the lysate constituents prior to activity assessment. Each data point represents a single experiment, and error bars represent S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9

    doi: 10.1074/jbc.M116.760579

    Figure Lengend Snippet: GS-5745 prevents activation of pro-MMP9 by ulcerative colitis lysates. A , colon tissue was lysed, and MMP9 was visualized by immunoblotting. The upper band (molecular mass, ∼92 kDa) was detected in diseased and healthy colon lysate, whereas the lower band (∼82 kDa) was detected in only the ulcerative colitis lysate. B , endogenous MMP9 activity from each colon lysate was assessed. A standard curve of quantitatively activated MMP9 was used to determine the equivalents of active MMP9 in ng/ml. C , incubation of recombinant pro-MMP9 with ulcerative colitis lysates results in MMP9 activation. The activated MMP9 was detected by the neoepitope antibody ( red ), whereas both pro- and active MMP9 are detected by the total MMP9 antibody ( green ). MMP9 activation is inhibited by GS-5745 but not a human IgG4 isotype control antibody. D , the activity of MMP9 in C was measured with the Biotrak MMP9 assay kit. GS-5745, but not the isotype control IgG4, blocks this proteolysis. MMP9 was isolated from the lysate constituents prior to activity assessment. Each data point represents a single experiment, and error bars represent S.D.

    Article Snippet: Reactions were incubated at 37 °C for various times, and pro- and active MMP9 were visualized by immunoblotting analysis using a rabbit monoclonal antibody raised against MMP9 (Abcam).

    Techniques: Activation Assay, Activity Assay, Incubation, Recombinant, Isolation

    Levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 measured by ELISA in the serum of one patient with MPS II. Each bar represents the mean ± SED from experiments performed in duplicate and are presented as % of values of age- and sex-matched

    Journal: JIMD Reports

    Article Title: Differential Expression of Matrix Metalloproteinases in the Serum of Patients with Mucopolysaccharidoses

    doi: 10.1007/8904_2011_58

    Figure Lengend Snippet: Levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 measured by ELISA in the serum of one patient with MPS II. Each bar represents the mean ± SED from experiments performed in duplicate and are presented as % of values of age- and sex-matched

    Article Snippet: Molecular size of bands displaying enzymatic activity were estimated by comparison to purified proMMP-2 (72 kDa), active MMP-2 (64 kDa), proMMP-9 (92 kDa), and active MMP-9 (78 kDa) (Anawa Trading, Wangen).

    Techniques: Enzyme-linked Immunosorbent Assay

    Concentration of circulating MMP-2 ( a ), MMP-9 ( b ), TIMP-1 ( c ), TIMP-2 ( d ), in the serum of patients with MPS III ( n = 5) and age- and sex-matched controls ( n = 5, for each MPS III patient) measured by ELISA. Each bar represents

    Journal: JIMD Reports

    Article Title: Differential Expression of Matrix Metalloproteinases in the Serum of Patients with Mucopolysaccharidoses

    doi: 10.1007/8904_2011_58

    Figure Lengend Snippet: Concentration of circulating MMP-2 ( a ), MMP-9 ( b ), TIMP-1 ( c ), TIMP-2 ( d ), in the serum of patients with MPS III ( n = 5) and age- and sex-matched controls ( n = 5, for each MPS III patient) measured by ELISA. Each bar represents

    Article Snippet: Molecular size of bands displaying enzymatic activity were estimated by comparison to purified proMMP-2 (72 kDa), active MMP-2 (64 kDa), proMMP-9 (92 kDa), and active MMP-9 (78 kDa) (Anawa Trading, Wangen).

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Enzyme replacement therapy alters protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the serum of patients with MPS VI. The protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured in the serum of a patient with MPS VI before the initiation

    Journal: JIMD Reports

    Article Title: Differential Expression of Matrix Metalloproteinases in the Serum of Patients with Mucopolysaccharidoses

    doi: 10.1007/8904_2011_58

    Figure Lengend Snippet: Enzyme replacement therapy alters protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the serum of patients with MPS VI. The protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured in the serum of a patient with MPS VI before the initiation

    Article Snippet: Molecular size of bands displaying enzymatic activity were estimated by comparison to purified proMMP-2 (72 kDa), active MMP-2 (64 kDa), proMMP-9 (92 kDa), and active MMP-9 (78 kDa) (Anawa Trading, Wangen).

    Techniques:

    Effect of mechanical stretch and IL-10 on MMP-9 release in epithelial cells and fibroblasts. Release of MMP-9 into the supernatant of E18–19 epithelial cells ( a ) and fibroblasts ( b ) exposed to 20% cyclic stretch for 48 h in the presence or absence

    Journal: Lung

    Article Title: Differential Expression of MMP-2 and -9 and their Inhibitors in Fetal Lung Cells Exposed to Mechanical Stretch: Regulation by IL-10

    doi: 10.1007/s00408-011-9310-7

    Figure Lengend Snippet: Effect of mechanical stretch and IL-10 on MMP-9 release in epithelial cells and fibroblasts. Release of MMP-9 into the supernatant of E18–19 epithelial cells ( a ) and fibroblasts ( b ) exposed to 20% cyclic stretch for 48 h in the presence or absence

    Article Snippet: The Fluorokine® E Enzyme Activity Assay for detection of Human Active MMP-9 (R & D Systems) was modified in the same manner as the Biotrak Activity Assay kit in order to detect the amount of active MMP-9 present in the supernatants.

    Techniques:

    Effect of mechanical stretch and IL-10 on TIMP-1 and MMP-9/TIMP-1 ratio. a E18–19 epithelial cells were harvested and subjected to 20% mechanical stretch for 48 h. Unstretched samples were used as controls. Levels of TIMP-1 present in cell lysates

    Journal: Lung

    Article Title: Differential Expression of MMP-2 and -9 and their Inhibitors in Fetal Lung Cells Exposed to Mechanical Stretch: Regulation by IL-10

    doi: 10.1007/s00408-011-9310-7

    Figure Lengend Snippet: Effect of mechanical stretch and IL-10 on TIMP-1 and MMP-9/TIMP-1 ratio. a E18–19 epithelial cells were harvested and subjected to 20% mechanical stretch for 48 h. Unstretched samples were used as controls. Levels of TIMP-1 present in cell lysates

    Article Snippet: The Fluorokine® E Enzyme Activity Assay for detection of Human Active MMP-9 (R & D Systems) was modified in the same manner as the Biotrak Activity Assay kit in order to detect the amount of active MMP-9 present in the supernatants.

    Techniques:

    Photographs from the epicardial surface (top) and endocardial surface (bottom) of hearts from MMP-9 gene promoter reporter mice showing regions of positive β-galactosidase staining (dark regions on lighter myocardium) at indicated time points after RF current-induced injury. Positive β-galactosidase staining was observed at 3 d post-RF injury and peaked at 7 d after MI. Scale grid at left represents a square with 2-mm sides.

    Journal: The FASEB Journal

    Article Title: Spatiotemporal induction of matrix metalloproteinase-9 transcription after discrete myocardial injury

    doi: 10.1096/fj.10-155531

    Figure Lengend Snippet: Photographs from the epicardial surface (top) and endocardial surface (bottom) of hearts from MMP-9 gene promoter reporter mice showing regions of positive β-galactosidase staining (dark regions on lighter myocardium) at indicated time points after RF current-induced injury. Positive β-galactosidase staining was observed at 3 d post-RF injury and peaked at 7 d after MI. Scale grid at left represents a square with 2-mm sides.

    Article Snippet: The cells were grown in plastic flasks to confluence (2.3×106 cells/ml), trypsinized, and transferred to reaction chambers to which either 0.41 μg/ml recombinant active MMP-9 (in DMEM, n =4, PF024; Oncogene Research Products) or DMEM (vehicle, n =5) was added.

    Techniques: Mouse Assay, Staining

    Left panels: representative photomicrograph of the RF-injured and border regions from hearts of MMP-9 promoter mice at 7 d after RF current stained using antibodies against MMP-9. Inset: higher-power micrograph from the highlighted area of the scar border. Positive staining for MMP-9 protein (brown) was localized at and around cells that elaborated β-galactosidase (blue). Right panels: Representative H E-stained serial sections from the same mouse heart showing increased cellularity at the border region. Inset: higher-power photomicrograph of the region shows disruption of the ECM within the region of myocardial injury. Scale bars = 100 μm (lower power); 20 μm (higher power).

    Journal: The FASEB Journal

    Article Title: Spatiotemporal induction of matrix metalloproteinase-9 transcription after discrete myocardial injury

    doi: 10.1096/fj.10-155531

    Figure Lengend Snippet: Left panels: representative photomicrograph of the RF-injured and border regions from hearts of MMP-9 promoter mice at 7 d after RF current stained using antibodies against MMP-9. Inset: higher-power micrograph from the highlighted area of the scar border. Positive staining for MMP-9 protein (brown) was localized at and around cells that elaborated β-galactosidase (blue). Right panels: Representative H E-stained serial sections from the same mouse heart showing increased cellularity at the border region. Inset: higher-power photomicrograph of the region shows disruption of the ECM within the region of myocardial injury. Scale bars = 100 μm (lower power); 20 μm (higher power).

    Article Snippet: The cells were grown in plastic flasks to confluence (2.3×106 cells/ml), trypsinized, and transferred to reaction chambers to which either 0.41 μg/ml recombinant active MMP-9 (in DMEM, n =4, PF024; Oncogene Research Products) or DMEM (vehicle, n =5) was added.

    Techniques: Mouse Assay, Staining

    Top panel: coincubation of recombinant Cx43 with increasing concentrations of recombinant, active MMP-9 did not cause a reduction in Cx43 immunoblotting. Middle panel: summary of immunoblot data. Bottom panel: Cx43 levels in HeLa cells transfected to express full length Cx43. There was no difference in Cx43 levels between HeLa cell cultures incubated with ( n =4) or without ( n =3) recombinant MMP-9. IOD, immunodetectable.

    Journal: The FASEB Journal

    Article Title: Spatiotemporal induction of matrix metalloproteinase-9 transcription after discrete myocardial injury

    doi: 10.1096/fj.10-155531

    Figure Lengend Snippet: Top panel: coincubation of recombinant Cx43 with increasing concentrations of recombinant, active MMP-9 did not cause a reduction in Cx43 immunoblotting. Middle panel: summary of immunoblot data. Bottom panel: Cx43 levels in HeLa cells transfected to express full length Cx43. There was no difference in Cx43 levels between HeLa cell cultures incubated with ( n =4) or without ( n =3) recombinant MMP-9. IOD, immunodetectable.

    Article Snippet: The cells were grown in plastic flasks to confluence (2.3×106 cells/ml), trypsinized, and transferred to reaction chambers to which either 0.41 μg/ml recombinant active MMP-9 (in DMEM, n =4, PF024; Oncogene Research Products) or DMEM (vehicle, n =5) was added.

    Techniques: Recombinant, Transfection, Incubation

    Full-length RECK and K23 inhibited MMP-9 secretion and activity. ( A ) Conditioned medium of A549 cells was incubated with 3 μg/ml of BSA ( B ), CKM, K123, K23 or full-length RECK at 37°C for 30 min. The MMP activity was studied as described in Materials and Methods. Results of three independent experiments were expressed as mean ± SE. MMP activity of the control group incubated with 3 μg/ml of BSA was defined as 100% (n = 3). * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The Kazal motifs of RECK protein inhibit MMP-9 secretion and activity and reduce metastasis of lung cancer cells in vitro and in vivo

    doi: 10.1111/j.1582-4934.2008.00215.x

    Figure Lengend Snippet: Full-length RECK and K23 inhibited MMP-9 secretion and activity. ( A ) Conditioned medium of A549 cells was incubated with 3 μg/ml of BSA ( B ), CKM, K123, K23 or full-length RECK at 37°C for 30 min. The MMP activity was studied as described in Materials and Methods. Results of three independent experiments were expressed as mean ± SE. MMP activity of the control group incubated with 3 μg/ml of BSA was defined as 100% (n = 3). * P

    Article Snippet: Active recombinant MMP-9 was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Activity Assay, Incubation

    K23 physically interacted with active MMP-9 and functioned as a non-competitive inhibitor. ( A ) Active recombinant MMP-9 protein (100 ng) was incubated with BSA ( B ) or K23 (500 ng) at 4°C for 2 hrs. Anti-MMP-9 antibody was added and the immunocomplex was precipitated by protein G-agarose beads. The binding of K23 to MMP-9 was detected by using anti-Hisx6 antibody and the blots were also probed with anti-MMP-9 antibody to confirm that the equal amount of MMP-9 was immunoprecipitated. ( B ) Active recombinant MMP-9 (5 ng) was mixed with various concentrations of a fluorescein-conjugated gelatin (DQ™ gelatin) and recombinant K23 protein (0, 41.7 or 125 nM). Experiments were performed similar to the procedures of MMP assay as described in Materials and methods.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The Kazal motifs of RECK protein inhibit MMP-9 secretion and activity and reduce metastasis of lung cancer cells in vitro and in vivo

    doi: 10.1111/j.1582-4934.2008.00215.x

    Figure Lengend Snippet: K23 physically interacted with active MMP-9 and functioned as a non-competitive inhibitor. ( A ) Active recombinant MMP-9 protein (100 ng) was incubated with BSA ( B ) or K23 (500 ng) at 4°C for 2 hrs. Anti-MMP-9 antibody was added and the immunocomplex was precipitated by protein G-agarose beads. The binding of K23 to MMP-9 was detected by using anti-Hisx6 antibody and the blots were also probed with anti-MMP-9 antibody to confirm that the equal amount of MMP-9 was immunoprecipitated. ( B ) Active recombinant MMP-9 (5 ng) was mixed with various concentrations of a fluorescein-conjugated gelatin (DQ™ gelatin) and recombinant K23 protein (0, 41.7 or 125 nM). Experiments were performed similar to the procedures of MMP assay as described in Materials and methods.

    Article Snippet: Active recombinant MMP-9 was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Recombinant, Incubation, Binding Assay, Immunoprecipitation, Mmp Assay

    R1R2 impairs the invading capacity of fibrocytes through the BM by reducing the proteolytic activity of MMP-9. (A, B) CD45 + , type I collagen + , and CXCR4 + fibrocytes isolated from bleomycin-treated lungs (14 days) were seeded in the upper chamber and treated with R1R2 or the scrambled peptide (1000 nM) for 24 h. CXCL12 (20 ng/mL) or 10% fetal bovine serum (FBS) were added in the bottom wells. CXCL12-induced fibrocyte invasion through Matrigel (A). Data are expressed as relative fluorescence units (RFU). The invasive capacities through Matrigel-coated inserts between R1R2- and scrambled peptide-treated fibrocytes are shown. Results were normalized for FBS-induced fibrocyte invasion. ** P

    Journal: PLoS ONE

    Article Title: R1R2 peptide ameliorates pulmonary fibrosis in mice through fibrocyte migration and differentiation

    doi: 10.1371/journal.pone.0185811

    Figure Lengend Snippet: R1R2 impairs the invading capacity of fibrocytes through the BM by reducing the proteolytic activity of MMP-9. (A, B) CD45 + , type I collagen + , and CXCR4 + fibrocytes isolated from bleomycin-treated lungs (14 days) were seeded in the upper chamber and treated with R1R2 or the scrambled peptide (1000 nM) for 24 h. CXCL12 (20 ng/mL) or 10% fetal bovine serum (FBS) were added in the bottom wells. CXCL12-induced fibrocyte invasion through Matrigel (A). Data are expressed as relative fluorescence units (RFU). The invasive capacities through Matrigel-coated inserts between R1R2- and scrambled peptide-treated fibrocytes are shown. Results were normalized for FBS-induced fibrocyte invasion. ** P

    Article Snippet: Mouse CXCL12 (SDF-1 beta, BioLegend, San Diego, CA, USA), human CXCL12 (SDF-1 beta, R & D, Minneapolis, MN, USA), human and mouse active MMP-9 (Abcam, Cambridge, MA, USA) and MMP-2 (R & D, Minneapolis, MN, USA) proteins, and DQ Gelatin® (Invitrogen, Waltham, MA, USA) were used for in vitro assays.

    Techniques: Activity Assay, Isolation, Fluorescence

    R1R2 increases CXCL12 degradation by MMP-9. CXCL12 degradation was determined by SDS-PAGE, followed by Coomassie blue staining. Lane 1: marker. Lane 2: MMP-9 (0.1 μg) was incubated with CXCL12 (2 μg) at room temperature for 3h. Lanes 3 and 4: MMP-9 was incubated with CXCL12 at room temperature for 3h with the scrambled peptide (100 μg) and R1R2 (100 μg), respectively. Lane 5 and 6: scrambled peptide and R1R2 maintained at room temperature for 3h. Lanes 7: MMP-9 was incubated with R1R2 at room temperature for 3h. The smear at the bottom of the gel indicated the slightly degraded products of proteins.

    Journal: PLoS ONE

    Article Title: R1R2 peptide ameliorates pulmonary fibrosis in mice through fibrocyte migration and differentiation

    doi: 10.1371/journal.pone.0185811

    Figure Lengend Snippet: R1R2 increases CXCL12 degradation by MMP-9. CXCL12 degradation was determined by SDS-PAGE, followed by Coomassie blue staining. Lane 1: marker. Lane 2: MMP-9 (0.1 μg) was incubated with CXCL12 (2 μg) at room temperature for 3h. Lanes 3 and 4: MMP-9 was incubated with CXCL12 at room temperature for 3h with the scrambled peptide (100 μg) and R1R2 (100 μg), respectively. Lane 5 and 6: scrambled peptide and R1R2 maintained at room temperature for 3h. Lanes 7: MMP-9 was incubated with R1R2 at room temperature for 3h. The smear at the bottom of the gel indicated the slightly degraded products of proteins.

    Article Snippet: Mouse CXCL12 (SDF-1 beta, BioLegend, San Diego, CA, USA), human CXCL12 (SDF-1 beta, R & D, Minneapolis, MN, USA), human and mouse active MMP-9 (Abcam, Cambridge, MA, USA) and MMP-2 (R & D, Minneapolis, MN, USA) proteins, and DQ Gelatin® (Invitrogen, Waltham, MA, USA) were used for in vitro assays.

    Techniques: SDS Page, Staining, Marker, Incubation

    R1R2 disables the proteolytic activity of MMP-9. (A) Equal amounts of R1R2, the scrambled peptide, or bovine serum albumin (BSA; 20 μg/mL) were coated onto 96-well plates; fibronectin (FN), collagen (Col), or phosphate-buffered saline (PBS) were added into the wells, and the interaction between the peptides was evaluated by measuring the absorbance at 405 nm. **** P

    Journal: PLoS ONE

    Article Title: R1R2 peptide ameliorates pulmonary fibrosis in mice through fibrocyte migration and differentiation

    doi: 10.1371/journal.pone.0185811

    Figure Lengend Snippet: R1R2 disables the proteolytic activity of MMP-9. (A) Equal amounts of R1R2, the scrambled peptide, or bovine serum albumin (BSA; 20 μg/mL) were coated onto 96-well plates; fibronectin (FN), collagen (Col), or phosphate-buffered saline (PBS) were added into the wells, and the interaction between the peptides was evaluated by measuring the absorbance at 405 nm. **** P

    Article Snippet: Mouse CXCL12 (SDF-1 beta, BioLegend, San Diego, CA, USA), human CXCL12 (SDF-1 beta, R & D, Minneapolis, MN, USA), human and mouse active MMP-9 (Abcam, Cambridge, MA, USA) and MMP-2 (R & D, Minneapolis, MN, USA) proteins, and DQ Gelatin® (Invitrogen, Waltham, MA, USA) were used for in vitro assays.

    Techniques: Activity Assay

    tPA-Mediated BM Reconstitution Requires Both Plg and MMP Activation Plg +/+ and Plg −/− mice (A–F) and MMP-9 +/+ and MMP-9 −/− mice (G–I) were injected with a single dose of 5-FU followed by daily injections with recombinant tPA or carrier. (A) Survival was assessed daily in Plg +/+ (n = 9 per group) and Plg −/− mice (n = 8 per group). WBC counts (B) and BM cell numbers per femur (C) were determined. Error bars represent SEM. (D) BM sections of Plg +/+ mice coinjection with or without tPA were stained for Plg 2 days after 5-FU treatment (brown staining). Plg staining was found along bone-lining cells. (E and F) BM myelogram was performed. Percentage of myeloid (E) and erythroid (F) BM cells are given. (G–I) BM cell numbers per femur (G), WBC counts (H), and survival (I) were assessed in MMP-9 +/+ and MMP-9 −/− mice (n = 12). *p

    Journal: Cell stem cell

    Article Title: The Plasminogen Fibrinolytic Pathway Is Required for Hematopoietic Regeneration

    doi: 10.1016/j.stem.2007.10.012

    Figure Lengend Snippet: tPA-Mediated BM Reconstitution Requires Both Plg and MMP Activation Plg +/+ and Plg −/− mice (A–F) and MMP-9 +/+ and MMP-9 −/− mice (G–I) were injected with a single dose of 5-FU followed by daily injections with recombinant tPA or carrier. (A) Survival was assessed daily in Plg +/+ (n = 9 per group) and Plg −/− mice (n = 8 per group). WBC counts (B) and BM cell numbers per femur (C) were determined. Error bars represent SEM. (D) BM sections of Plg +/+ mice coinjection with or without tPA were stained for Plg 2 days after 5-FU treatment (brown staining). Plg staining was found along bone-lining cells. (E and F) BM myelogram was performed. Percentage of myeloid (E) and erythroid (F) BM cells are given. (G–I) BM cell numbers per femur (G), WBC counts (H), and survival (I) were assessed in MMP-9 +/+ and MMP-9 −/− mice (n = 12). *p

    Article Snippet: Active MMP-9 was analyzed using the MMP-9 Activity Assay Biotrak System ELISA (Amersham Biosciences, UK).

    Techniques: Activation Assay, Mouse Assay, Injection, Recombinant, Staining

    MMP Activation and KitL Release Are Impaired in Plg −/− Mice after Myelosuppression, Resulting in BM Recovery Failure (A–D) Plg +/+ and Plg −/− mice were injected with a single dose of 5-FU i.v. (A) BM cells (three mice per time point) were cultured in serum-free medium overnight. Cell supernatants were assayed for proMMP-9 (103 kDa), active MMP-9 (86 kDa), proMMP-2 (72 kDa), and active MMP-2 (62 kDa) by gelatin zymography. Error bars represent standard deviation. (B) Immunohistochemistry of BM sections 9 days after 5-FU injection for proMMP-9 with positive staining in the BM stromal compartment of Plg +/+ , but less in the BM stromal compartment of Plg −/− mice. Magnification ×200. (C and D) Plasma obtained from peripheral blood (PB) was assayed for active MMP-9 (C) or KitL (D) by ELISA (p

    Journal: Cell stem cell

    Article Title: The Plasminogen Fibrinolytic Pathway Is Required for Hematopoietic Regeneration

    doi: 10.1016/j.stem.2007.10.012

    Figure Lengend Snippet: MMP Activation and KitL Release Are Impaired in Plg −/− Mice after Myelosuppression, Resulting in BM Recovery Failure (A–D) Plg +/+ and Plg −/− mice were injected with a single dose of 5-FU i.v. (A) BM cells (three mice per time point) were cultured in serum-free medium overnight. Cell supernatants were assayed for proMMP-9 (103 kDa), active MMP-9 (86 kDa), proMMP-2 (72 kDa), and active MMP-2 (62 kDa) by gelatin zymography. Error bars represent standard deviation. (B) Immunohistochemistry of BM sections 9 days after 5-FU injection for proMMP-9 with positive staining in the BM stromal compartment of Plg +/+ , but less in the BM stromal compartment of Plg −/− mice. Magnification ×200. (C and D) Plasma obtained from peripheral blood (PB) was assayed for active MMP-9 (C) or KitL (D) by ELISA (p

    Article Snippet: Active MMP-9 was analyzed using the MMP-9 Activity Assay Biotrak System ELISA (Amersham Biosciences, UK).

    Techniques: Activation Assay, Mouse Assay, Injection, Cell Culture, Zymography, Standard Deviation, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

    tPA-Mediated MMP Activation Releases KitL from Stromal Cells (A) RT-PCR for tPA and Plg in liver (sample 1; positive control for Plg), 4-week-old BM stroma of Plg +/+ mice (sample 2) and of Plg −/− mice (sample 3), MS-5 cells (sample 4), freshly isolated BM-derived lin− cells from Plg +/+ mice (sample 5), or Plg −/− mice (sample 6), as well as freshly isolated BM-derived lin+ cells from Plg +/+ mice (sample 7) or Plg −/− mice (sample 8). Agarose gel of one representative experiment. (B) Immunohistochemistry for tPA in BM sections of Plg +/+ mice under steady-state conditions (magnification ×200). (Insert) Vessel stained positive for tPA. (C–F) MS-5 cells (C) or lin+ BMMCs from Plg +/+ mice (D) were cultured overnight with/without tPA under serum-free conditions. Supernatants were analyzed by zymography for MMP-2 and MMP-9. Error bars represent standard deviation. Immunohistochemistry for Plg (E) and MMP-9 (F) in BM sections of Plg +/+ mice 3 days after starting tPA treatment (magnification ×200). (G and H) Confluent MS-5 stromal cells (G) or Plg +/+ primary BM stroma cells (H) were cultured overnight in serum-free medium (n = 3) in the presence of recombinant tPA, recombinant PAI-1, and MPI (CGS 27023A) with or without tPA. Supernatants were collected and analyzed for KitL by ELISA (n = 3, p

    Journal: Cell stem cell

    Article Title: The Plasminogen Fibrinolytic Pathway Is Required for Hematopoietic Regeneration

    doi: 10.1016/j.stem.2007.10.012

    Figure Lengend Snippet: tPA-Mediated MMP Activation Releases KitL from Stromal Cells (A) RT-PCR for tPA and Plg in liver (sample 1; positive control for Plg), 4-week-old BM stroma of Plg +/+ mice (sample 2) and of Plg −/− mice (sample 3), MS-5 cells (sample 4), freshly isolated BM-derived lin− cells from Plg +/+ mice (sample 5), or Plg −/− mice (sample 6), as well as freshly isolated BM-derived lin+ cells from Plg +/+ mice (sample 7) or Plg −/− mice (sample 8). Agarose gel of one representative experiment. (B) Immunohistochemistry for tPA in BM sections of Plg +/+ mice under steady-state conditions (magnification ×200). (Insert) Vessel stained positive for tPA. (C–F) MS-5 cells (C) or lin+ BMMCs from Plg +/+ mice (D) were cultured overnight with/without tPA under serum-free conditions. Supernatants were analyzed by zymography for MMP-2 and MMP-9. Error bars represent standard deviation. Immunohistochemistry for Plg (E) and MMP-9 (F) in BM sections of Plg +/+ mice 3 days after starting tPA treatment (magnification ×200). (G and H) Confluent MS-5 stromal cells (G) or Plg +/+ primary BM stroma cells (H) were cultured overnight in serum-free medium (n = 3) in the presence of recombinant tPA, recombinant PAI-1, and MPI (CGS 27023A) with or without tPA. Supernatants were collected and analyzed for KitL by ELISA (n = 3, p

    Article Snippet: Active MMP-9 was analyzed using the MMP-9 Activity Assay Biotrak System ELISA (Amersham Biosciences, UK).

    Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Mouse Assay, Mass Spectrometry, Isolation, Derivative Assay, Agarose Gel Electrophoresis, Immunohistochemistry, Staining, Cell Culture, Zymography, Standard Deviation, Recombinant, Enzyme-linked Immunosorbent Assay

    tPA Promotes HSPC Proliferation in KitL-Competent Mice (A) Plg-deficient, MMP-9-deficient, and littermate mice were injected with recombinant tPA i.p. from day 0–2 (short-term effect), and BM cells were counted. BM cells of Plg +/+ and Plg −/− cells were analyzed in a CFU-S assay ([B], n = 9), LTC-IC assay ([C], n = 9), and by FACS for the presence of KSL cells ([D], n = 9). (E) Plg +/+ and Plg −/− mice were injected with recombinant tPA from day 0–5 (long-term effect), and BM cells were counted (n = 9). (F and G) Sl/Sl d and WBB6F1 mice were treated with/without tPA (n = 5). BM cell numbers per femur (F) and the number of CFU-S were determined (G). (H) Mouse PB cell donor contribution transplantation of BM cells from Plg +/+ and Plg −/− BM CD45.2+ cells at limiting cell dilutions into CD45.1+ recipient mice (n = 9 per cell concentration); *p

    Journal: Cell stem cell

    Article Title: The Plasminogen Fibrinolytic Pathway Is Required for Hematopoietic Regeneration

    doi: 10.1016/j.stem.2007.10.012

    Figure Lengend Snippet: tPA Promotes HSPC Proliferation in KitL-Competent Mice (A) Plg-deficient, MMP-9-deficient, and littermate mice were injected with recombinant tPA i.p. from day 0–2 (short-term effect), and BM cells were counted. BM cells of Plg +/+ and Plg −/− cells were analyzed in a CFU-S assay ([B], n = 9), LTC-IC assay ([C], n = 9), and by FACS for the presence of KSL cells ([D], n = 9). (E) Plg +/+ and Plg −/− mice were injected with recombinant tPA from day 0–5 (long-term effect), and BM cells were counted (n = 9). (F and G) Sl/Sl d and WBB6F1 mice were treated with/without tPA (n = 5). BM cell numbers per femur (F) and the number of CFU-S were determined (G). (H) Mouse PB cell donor contribution transplantation of BM cells from Plg +/+ and Plg −/− BM CD45.2+ cells at limiting cell dilutions into CD45.1+ recipient mice (n = 9 per cell concentration); *p

    Article Snippet: Active MMP-9 was analyzed using the MMP-9 Activity Assay Biotrak System ELISA (Amersham Biosciences, UK).

    Techniques: Mouse Assay, Injection, Recombinant, FACS, Transplantation Assay, Concentration Assay

    The effect of maternal hypoxia on the activity of MMP-2 and MMP-9. Hearts were isolated from E21 and PD7 rats in the control and hypoxic groups. The activities of MMP-2 and MMP-9 were determined by gelatin zymography. Clear bands indicate positive MMP

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Maternal hypoxia alters matrix metalloproteinase expression patterns and causes cardiac remodeling in fetal and neonatal rats

    doi: 10.1152/ajpheart.00356.2011

    Figure Lengend Snippet: The effect of maternal hypoxia on the activity of MMP-2 and MMP-9. Hearts were isolated from E21 and PD7 rats in the control and hypoxic groups. The activities of MMP-2 and MMP-9 were determined by gelatin zymography. Clear bands indicate positive MMP

    Article Snippet: Mouse active MMP-9 (Chemicon, Temecular, CA) was used as a positive control.

    Techniques: Activity Assay, Isolation, Zymography

    MMP-9 activation of endothelial cells. (A) Effect of stimulation with MMP-9 on HUVEC cell activation (pERK); n = 4. (B) Effect of stimulation with MMP-9 on HUVEC cell apoptosis (caspase-3 activity; p

    Journal: PLoS ONE

    Article Title: Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    doi: 10.1371/journal.pone.0171427

    Figure Lengend Snippet: MMP-9 activation of endothelial cells. (A) Effect of stimulation with MMP-9 on HUVEC cell activation (pERK); n = 4. (B) Effect of stimulation with MMP-9 on HUVEC cell apoptosis (caspase-3 activity; p

    Article Snippet: All of aortic leaflets and normal aortic tissues specimens were evaluated for the expression of cleaved caspase 3 (rabbit anti-human cleaved caspase 3, Cell Signaling), PAR-1 (goat anti-human PAR-1, Santa Cruz Biotechnology), and active MMP-9 (mouse anti-human active MMP-9, Santa Cruz Biotechnology).

    Techniques: Activation Assay, Activity Assay

    MMP-9 interaction with endothelial cell PAR-1. (A) Activation of PAR-1 by MMP-9 (pseudo color green); n = 3. (B) Expression of pERK in HUVEC cells by MMP-9 in presence and absence of anti-PAR-1; n = 3. (C). Expression of MMP-9 and PAR-1 in aortic leaflets from patients with atherosclerosis; n = 3.

    Journal: PLoS ONE

    Article Title: Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    doi: 10.1371/journal.pone.0171427

    Figure Lengend Snippet: MMP-9 interaction with endothelial cell PAR-1. (A) Activation of PAR-1 by MMP-9 (pseudo color green); n = 3. (B) Expression of pERK in HUVEC cells by MMP-9 in presence and absence of anti-PAR-1; n = 3. (C). Expression of MMP-9 and PAR-1 in aortic leaflets from patients with atherosclerosis; n = 3.

    Article Snippet: All of aortic leaflets and normal aortic tissues specimens were evaluated for the expression of cleaved caspase 3 (rabbit anti-human cleaved caspase 3, Cell Signaling), PAR-1 (goat anti-human PAR-1, Santa Cruz Biotechnology), and active MMP-9 (mouse anti-human active MMP-9, Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Level of MMP-9 (total and endogenous active) in plasma of WD + SHS exposed ApoE -/- mice. (A) Total MMP-9 in plasma at 7 weeks and endogenous active MMP-9 at 13 weeks exposure. Values are shown as mean ± STD, † p

    Journal: PLoS ONE

    Article Title: Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

    doi: 10.1371/journal.pone.0171427

    Figure Lengend Snippet: Level of MMP-9 (total and endogenous active) in plasma of WD + SHS exposed ApoE -/- mice. (A) Total MMP-9 in plasma at 7 weeks and endogenous active MMP-9 at 13 weeks exposure. Values are shown as mean ± STD, † p

    Article Snippet: All of aortic leaflets and normal aortic tissues specimens were evaluated for the expression of cleaved caspase 3 (rabbit anti-human cleaved caspase 3, Cell Signaling), PAR-1 (goat anti-human PAR-1, Santa Cruz Biotechnology), and active MMP-9 (mouse anti-human active MMP-9, Santa Cruz Biotechnology).

    Techniques: Mouse Assay

    Age-dependent blood-brain barrier breakdown and elevated cyclophilin A and active MMP-9 levels in cerebrospinal fluid (CSF) of cognitively normal APOE4 carriers

    Journal: JAMA neurology

    Article Title: Relationship Between Cyclophilin A Levels and Matrix Metalloproteinase 9 Activity in Cerebrospinal Fluid of Cognitively Normal Apolipoprotein E4 Carriers and Blood-Brain Barrier Breakdown

    doi: 10.1001/jamaneurol.2013.3841

    Figure Lengend Snippet: Age-dependent blood-brain barrier breakdown and elevated cyclophilin A and active MMP-9 levels in cerebrospinal fluid (CSF) of cognitively normal APOE4 carriers

    Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were used to determine levels of CypA (Cat. No. sE90979Hu, USCN Life Science, Houston, TX), active MMP-9 (Cat. No. 72017, AnaSpec, Fremont, CA) and albumin (Cat. No. E-80AL, Immunology Consultant Laboratories, Portland, OR).

    Techniques:

    Mechanism of oxygen-glucose deprivation (OGD)-preconditioned microglial transplantation after cerebral ischemia. ( A ) The schema of angiogenesis after cerebral ischemia. The ischemic core is defined as the MAP2-immunonegative ischemic cortex. Angiogenesis (red lines) is slightly activated at the border area within the ischemic core, which we define as the “angiogenesis-positive core” (dark grey), by cell therapy. Thus, the MAP2-negative ischemic core consists of the angiogenesis-positive core and angiogenesis-negative core (black), which develops irreversible change. ( B ) The schema of angiogenesis and axonal outgrowth (regeneration, green cells; neurons) by OGD-preconditioned microglial transplantation (blue cells) after cerebral ischemia. OGD-preconditioned microglial transplantation markedly activated angiogenesis (red lines) at the angiogenesis-positive core (ischemic border area). In addition, the decrease in the expression of chondroitin sulphate proteoglycan (CSPG, grey and diagonal area), which is known to be an axonal outgrowth inhibitor, was observed in the ischemic penumbra after cerebral ischemia. An axonal outgrowth of neuronal cells (green) by OGD-preconditioned microglial transplantation was observed in the ischemic penumbra. ( C ) A diagram of therapeutic effects of OGD-preconditioned microglial transplantation after cerebral ischemia. Transplanted OGD-preconditioned microglia directly secreted vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and matrix metalloproteinase-9 (MMP-9). These factors may also be associated with changes in resident native endothelial cells, astrocytes, pericytes, neurons, and microglia caused by the paracrine action of OGD-preconditioned M2 microglia. These factors directly prompt angiogenesis in the angiogenesis-positive core (ischemic border area). MMP-9 from microglia might degrade the axonal outgrowth inhibitor CSPG. Angiogenesis might facilitate the induction of axonal outgrowth. In addition, VEGF, TGF-β, and MMP-9 also might directly induce axonal outgrowth.

    Journal: Scientific Reports

    Article Title: Microglia preconditioned by oxygen-glucose deprivation promote functional recovery in ischemic rats

    doi: 10.1038/srep42582

    Figure Lengend Snippet: Mechanism of oxygen-glucose deprivation (OGD)-preconditioned microglial transplantation after cerebral ischemia. ( A ) The schema of angiogenesis after cerebral ischemia. The ischemic core is defined as the MAP2-immunonegative ischemic cortex. Angiogenesis (red lines) is slightly activated at the border area within the ischemic core, which we define as the “angiogenesis-positive core” (dark grey), by cell therapy. Thus, the MAP2-negative ischemic core consists of the angiogenesis-positive core and angiogenesis-negative core (black), which develops irreversible change. ( B ) The schema of angiogenesis and axonal outgrowth (regeneration, green cells; neurons) by OGD-preconditioned microglial transplantation (blue cells) after cerebral ischemia. OGD-preconditioned microglial transplantation markedly activated angiogenesis (red lines) at the angiogenesis-positive core (ischemic border area). In addition, the decrease in the expression of chondroitin sulphate proteoglycan (CSPG, grey and diagonal area), which is known to be an axonal outgrowth inhibitor, was observed in the ischemic penumbra after cerebral ischemia. An axonal outgrowth of neuronal cells (green) by OGD-preconditioned microglial transplantation was observed in the ischemic penumbra. ( C ) A diagram of therapeutic effects of OGD-preconditioned microglial transplantation after cerebral ischemia. Transplanted OGD-preconditioned microglia directly secreted vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and matrix metalloproteinase-9 (MMP-9). These factors may also be associated with changes in resident native endothelial cells, astrocytes, pericytes, neurons, and microglia caused by the paracrine action of OGD-preconditioned M2 microglia. These factors directly prompt angiogenesis in the angiogenesis-positive core (ischemic border area). MMP-9 from microglia might degrade the axonal outgrowth inhibitor CSPG. Angiogenesis might facilitate the induction of axonal outgrowth. In addition, VEGF, TGF-β, and MMP-9 also might directly induce axonal outgrowth.

    Article Snippet: Active MMP-9 assay The Fluorimetric SensoLyteTM 520 (AnaSpec Corp. San Jose, CA, USA) was used to quantify the specific enzymatic activity of active MMP-9 using a fluorescence resonance energy transfer (FRET) peptide containing a fluorescent donor and quenching acceptor .

    Techniques: Transplantation Assay, Expressing

    Transplantation of OGD-preconditioned microglia promotes expression of remodelling factors at 28 days after cerebral ischemia. ( A–C ) Representative figures and the relative signal intensities of vascular endothelial growth factor (VEGF) ( A ), matrix metalloproteinase-9 (MMP-9) ( B ), and transforming growth factor-β (TGF-β) ( C ) from cerebral cortices of the no cell control group and the microglia or astrocyte transplanted groups at 28 days after cerebral ischemia. VEGF ( A ), MMP-9 ( B ), and TGF-β ( C ) (red)/MAP2 (green)/DAPI (blue) triple labelling of cerebral cortices in the border between the ischemic core and penumbra at 28 days after reperfusion as examined by confocal microscopy. A secondary-only antibody control confirms its specificity. Scale bars, 15 μm. Bar graphs represent relative signal intensities of ischemic brain samples compared with those of no cell control samples (N = 21–28). **P

    Journal: Scientific Reports

    Article Title: Microglia preconditioned by oxygen-glucose deprivation promote functional recovery in ischemic rats

    doi: 10.1038/srep42582

    Figure Lengend Snippet: Transplantation of OGD-preconditioned microglia promotes expression of remodelling factors at 28 days after cerebral ischemia. ( A–C ) Representative figures and the relative signal intensities of vascular endothelial growth factor (VEGF) ( A ), matrix metalloproteinase-9 (MMP-9) ( B ), and transforming growth factor-β (TGF-β) ( C ) from cerebral cortices of the no cell control group and the microglia or astrocyte transplanted groups at 28 days after cerebral ischemia. VEGF ( A ), MMP-9 ( B ), and TGF-β ( C ) (red)/MAP2 (green)/DAPI (blue) triple labelling of cerebral cortices in the border between the ischemic core and penumbra at 28 days after reperfusion as examined by confocal microscopy. A secondary-only antibody control confirms its specificity. Scale bars, 15 μm. Bar graphs represent relative signal intensities of ischemic brain samples compared with those of no cell control samples (N = 21–28). **P

    Article Snippet: Active MMP-9 assay The Fluorimetric SensoLyteTM 520 (AnaSpec Corp. San Jose, CA, USA) was used to quantify the specific enzymatic activity of active MMP-9 using a fluorescence resonance energy transfer (FRET) peptide containing a fluorescent donor and quenching acceptor .

    Techniques: Transplantation Assay, Expressing, Confocal Microscopy

    TB patients carrying the two-locus genotype -2518 MCP-1 GG -1607 MMP-1 2G/2G have the highest serum levels of MCP-1 and the highest plasma levels of MMP-1 and MMP9 Values are shown as medians (horizontal lines), the 25th and 75th percentiles (boxes), and ranges (whiskers). Legends in the x-axis mean: 1 = two-locus genotype A/- 1G/-; 2 = two-locus genotype A/- 2G/2G; 3 = two-locus genotype GG 1G/-; 4 = two-locus genotype GG 2G/2G ). Section A: Distribution of serum MCP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the serum levels of MCP-1 across genotypes (ANOVA F = 16.31; p = 0.0001). Section B: Distribution of plasma MMP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-1 across genotypes (ANOVA F = 5.76; p = 0.001). Section C: Distribution of plasma MMP-9 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-9 across genotypes (ANOVA F = 8.25; p = 0.0001). Comparison of means and standard deviations by genotypes are shown at the right of the whiskers and box figures. The p-values are based on the Bonferroni least significant difference test.

    Journal: Genes and immunity

    Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

    doi: 10.1038/gene.2012.39

    Figure Lengend Snippet: TB patients carrying the two-locus genotype -2518 MCP-1 GG -1607 MMP-1 2G/2G have the highest serum levels of MCP-1 and the highest plasma levels of MMP-1 and MMP9 Values are shown as medians (horizontal lines), the 25th and 75th percentiles (boxes), and ranges (whiskers). Legends in the x-axis mean: 1 = two-locus genotype A/- 1G/-; 2 = two-locus genotype A/- 2G/2G; 3 = two-locus genotype GG 1G/-; 4 = two-locus genotype GG 2G/2G ). Section A: Distribution of serum MCP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the serum levels of MCP-1 across genotypes (ANOVA F = 16.31; p = 0.0001). Section B: Distribution of plasma MMP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-1 across genotypes (ANOVA F = 5.76; p = 0.001). Section C: Distribution of plasma MMP-9 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-9 across genotypes (ANOVA F = 8.25; p = 0.0001). Comparison of means and standard deviations by genotypes are shown at the right of the whiskers and box figures. The p-values are based on the Bonferroni least significant difference test.

    Article Snippet: MMP-1 and MMP-9 were measured using Fluorokine E, Human Active MMP-1 or Fluorokine E, and Human Active MMP-9 kits (R & D Systems).

    Techniques:

    CCR2 RS504393 and MMP-1 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide inhibitors modulate the expression of MCP-1, MMP-1, and MMP-9 and, to a lesser extent the expression of TIMPs inTHP-1 monocytic cells stimulated by sonicated H37Rv M. tuberculosis We measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. Panel 1 , the effect of 10 μM concentration of CCR2 RS504393 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. CCR2 RS504393 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p

    Journal: Genes and immunity

    Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

    doi: 10.1038/gene.2012.39

    Figure Lengend Snippet: CCR2 RS504393 and MMP-1 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide inhibitors modulate the expression of MCP-1, MMP-1, and MMP-9 and, to a lesser extent the expression of TIMPs inTHP-1 monocytic cells stimulated by sonicated H37Rv M. tuberculosis We measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. Panel 1 , the effect of 10 μM concentration of CCR2 RS504393 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. CCR2 RS504393 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p

    Article Snippet: MMP-1 and MMP-9 were measured using Fluorokine E, Human Active MMP-1 or Fluorokine E, and Human Active MMP-9 kits (R & D Systems).

    Techniques: Expressing, Sonication, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro

    PAR-1 inhibitor SCH79797 regulates the expression of MCP-1, MMP-1, and MMP-9 in THP-1 monocytic cells stimulated by sonicated H37Rv M. tuberculosis and THP-1 cells stimulated by sonicated H37Rv M. tuberculosis in the presence of exogenous purified human MMP-1 Panel 1, we measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. The effect of 50 nM concentration of SCH79797 PAR-1 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl incomplete RPMI. PAR-1 SCH79797 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p

    Journal: Genes and immunity

    Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

    doi: 10.1038/gene.2012.39

    Figure Lengend Snippet: PAR-1 inhibitor SCH79797 regulates the expression of MCP-1, MMP-1, and MMP-9 in THP-1 monocytic cells stimulated by sonicated H37Rv M. tuberculosis and THP-1 cells stimulated by sonicated H37Rv M. tuberculosis in the presence of exogenous purified human MMP-1 Panel 1, we measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. The effect of 50 nM concentration of SCH79797 PAR-1 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl incomplete RPMI. PAR-1 SCH79797 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p

    Article Snippet: MMP-1 and MMP-9 were measured using Fluorokine E, Human Active MMP-1 or Fluorokine E, and Human Active MMP-9 kits (R & D Systems).

    Techniques: Expressing, Sonication, Purification, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro

    TB patients carrying the two-locus genotype -2518 MCP-1 GG -1607 MMP-1 2G/2G express MCP-1, MMP-1, and MMP9 We used double IHC analysis of MCP-1 or MMP-1 or MMP-9 and CD68 in paraffin-embedded diseased lung from six TB patients who underwent surgery to remove damaged tissue. We are showing representative data. A: negative control (irrelevant antibodies isotype control); B: MCP-1 (blue) and CD68 (red); C: MMP-1 (blue) and CD68 (light red); D: MMP-9 (blue) and CD68 (light red). Images were acquired at 200X total magnification. IHC analysis shows granulomas with CD68-positive cells (macrophages) expressing copious amounts of MCP-1, MMP-1, and MMP-9.

    Journal: Genes and immunity

    Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

    doi: 10.1038/gene.2012.39

    Figure Lengend Snippet: TB patients carrying the two-locus genotype -2518 MCP-1 GG -1607 MMP-1 2G/2G express MCP-1, MMP-1, and MMP9 We used double IHC analysis of MCP-1 or MMP-1 or MMP-9 and CD68 in paraffin-embedded diseased lung from six TB patients who underwent surgery to remove damaged tissue. We are showing representative data. A: negative control (irrelevant antibodies isotype control); B: MCP-1 (blue) and CD68 (red); C: MMP-1 (blue) and CD68 (light red); D: MMP-9 (blue) and CD68 (light red). Images were acquired at 200X total magnification. IHC analysis shows granulomas with CD68-positive cells (macrophages) expressing copious amounts of MCP-1, MMP-1, and MMP-9.

    Article Snippet: MMP-1 and MMP-9 were measured using Fluorokine E, Human Active MMP-1 or Fluorokine E, and Human Active MMP-9 kits (R & D Systems).

    Techniques: Immunohistochemistry, Negative Control, Expressing

    GC frass and a TLR2 agonist induced MMP-9 release from neutrophils. A. HL-60 cells were treated with PBS, GC frass, Pam-3-Cys (1 μg/ml) with and without pretreatment with lipoprotein lipase (1000U/mg) for 18 h. Supernatants were harvested, clarified,

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: GC frass and a TLR2 agonist induced MMP-9 release from neutrophils. A. HL-60 cells were treated with PBS, GC frass, Pam-3-Cys (1 μg/ml) with and without pretreatment with lipoprotein lipase (1000U/mg) for 18 h. Supernatants were harvested, clarified,

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques:

    MMP-9 levels were increased in BAL fluid of GC frass-treated mice. Balb/c mice were given a single intratracheal inhalation of PBS or GC frass and BAL fluid was harvested 18h later. MMP-9 activity was measured in the BAL fluid as determined by gelatin

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: MMP-9 levels were increased in BAL fluid of GC frass-treated mice. Balb/c mice were given a single intratracheal inhalation of PBS or GC frass and BAL fluid was harvested 18h later. MMP-9 activity was measured in the BAL fluid as determined by gelatin

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques: Mouse Assay, Activity Assay

    Neutrophil recruitment directly affected MMP-9 release into the airways. Balb/c mice were given a single injection of control Ab or RB65-8C5 (100 μg/mouse) 24h prior to a single intratracheal inhalation of PBS (40μl) or GC frass (40μg/40μl).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: Neutrophil recruitment directly affected MMP-9 release into the airways. Balb/c mice were given a single injection of control Ab or RB65-8C5 (100 μg/mouse) 24h prior to a single intratracheal inhalation of PBS (40μl) or GC frass (40μg/40μl).

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques: Mouse Assay, Injection

    Induced allergic inflammation is increased in MMP-9-deficient mice

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: Induced allergic inflammation is increased in MMP-9-deficient mice

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques: Mouse Assay

    MMP-9 levels in BAL fluid

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: MMP-9 levels in BAL fluid

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques:

    Neutrophil accumulation and MMP-9 levels in the airways of TLR2-deficient mice. Wild type (C57Bl/6) and TLR2-deficient mice were given a single intratracheal inhalation of PBS (40 μl) or GC frass (40 μg/40 μl) and 3 h later BAL

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: Neutrophil accumulation and MMP-9 levels in the airways of TLR2-deficient mice. Wild type (C57Bl/6) and TLR2-deficient mice were given a single intratracheal inhalation of PBS (40 μl) or GC frass (40 μg/40 μl) and 3 h later BAL

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques: Mouse Assay

    GC frass-induced experimental allergic asthma in MMP-9-deficient mice. Wild type (FVB) or MMP-9-deficient mice were challenged by intratracheal inhalation on day 0, 7, and 14 with PBS (40 μl) or GC frass (40 μg/40 μl). On day 19,

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: GC frass-induced experimental allergic asthma in MMP-9-deficient mice. Wild type (FVB) or MMP-9-deficient mice were challenged by intratracheal inhalation on day 0, 7, and 14 with PBS (40 μl) or GC frass (40 μg/40 μl). On day 19,

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques: Mouse Assay

    Histological assessment of lung sections from PBS or GC frass exposed wild type or MMP-9-deficient mice. Periodic Acid Schiff (PAS) staining of sectioned lungs from wild type (FVB) PBS (A) or GC frass (B) treated mice and MMP-9-deficient PBS (C) or GC

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A TLR2 Agonist in German Cockroach Frass Activates MMP-9 Release and is Protective Against Allergic Inflammation in Mice

    doi: 10.4049/jimmunol.0900838

    Figure Lengend Snippet: Histological assessment of lung sections from PBS or GC frass exposed wild type or MMP-9-deficient mice. Periodic Acid Schiff (PAS) staining of sectioned lungs from wild type (FVB) PBS (A) or GC frass (B) treated mice and MMP-9-deficient PBS (C) or GC

    Article Snippet: Mature (active) MMP-9 was determined by Western blot under reducing conditions (R & D Systems).

    Techniques: Mouse Assay, Staining

    The binding of CLU with MMP-9 or VEGF A. the binding of CLU and MMP-9 or VEGF was determined using immunoprecipitation and Western-blotting. 5-8F cells and 6-10B cells with DNP treatment were immunoprecipitated using anti-CLU body, and MMP-9 or VEGF was detected in the immunocomplexs by Western-blotting (a). MMP-9 or VEGF was respectively immunoprecipitated in the indicated cells using MMP-9 (b) or VEGF (c) antibody, and CLU was detected using Western-blotting. IP: immunoprecipitation; WB: Western-blotting. B. CLU co-localizes and binds with MMP-9. C. CLU co-localizes and binds to VEGF. DNP-treated or untreated 6-10B cell were fixed with paraformaldehyde, stained for CLU (green) and MMP-9 or VEGF (red), and then visualized by immunofluorescence microscopy. The localization and binding of CLU and MMP-9 or VEGF were indicated. Original magnification, ×1000. Scale bar, 50 μm. Arrow, CLU binding to MMP-9 or VEGF.

    Journal: Oncotarget

    Article Title: Clusterin induced by N,N′-Dinitrosopiperazine is involved in nasopharyngeal carcinoma metastasis

    doi: 10.18632/oncotarget.6750

    Figure Lengend Snippet: The binding of CLU with MMP-9 or VEGF A. the binding of CLU and MMP-9 or VEGF was determined using immunoprecipitation and Western-blotting. 5-8F cells and 6-10B cells with DNP treatment were immunoprecipitated using anti-CLU body, and MMP-9 or VEGF was detected in the immunocomplexs by Western-blotting (a). MMP-9 or VEGF was respectively immunoprecipitated in the indicated cells using MMP-9 (b) or VEGF (c) antibody, and CLU was detected using Western-blotting. IP: immunoprecipitation; WB: Western-blotting. B. CLU co-localizes and binds with MMP-9. C. CLU co-localizes and binds to VEGF. DNP-treated or untreated 6-10B cell were fixed with paraformaldehyde, stained for CLU (green) and MMP-9 or VEGF (red), and then visualized by immunofluorescence microscopy. The localization and binding of CLU and MMP-9 or VEGF were indicated. Original magnification, ×1000. Scale bar, 50 μm. Arrow, CLU binding to MMP-9 or VEGF.

    Article Snippet: Enzymatic activity assay Activities of MMP-9 in the cells and NPC biopsy tissues were assayed using Cell or Tissue Active MMP-9 Fluorescent Assay Kit (Genmed Scientifics Inc. USA) following the manufacturer's instructions.

    Techniques: Binding Assay, Immunoprecipitation, Western Blot, Staining, Immunofluorescence, Microscopy

    Schematic illustration of DNP-promoted nasopharyngeal carcinoma metastasis through CLU DNP induces CLU expression, binds to and increases MMP-9 and VEGF expression, increases motility and invasion of NPC cancer, and promotes NPC metastasis.

    Journal: Oncotarget

    Article Title: Clusterin induced by N,N′-Dinitrosopiperazine is involved in nasopharyngeal carcinoma metastasis

    doi: 10.18632/oncotarget.6750

    Figure Lengend Snippet: Schematic illustration of DNP-promoted nasopharyngeal carcinoma metastasis through CLU DNP induces CLU expression, binds to and increases MMP-9 and VEGF expression, increases motility and invasion of NPC cancer, and promotes NPC metastasis.

    Article Snippet: Enzymatic activity assay Activities of MMP-9 in the cells and NPC biopsy tissues were assayed using Cell or Tissue Active MMP-9 Fluorescent Assay Kit (Genmed Scientifics Inc. USA) following the manufacturer's instructions.

    Techniques: Expressing

    DNP induces expressions of CLU, MMP-9 and VEGF A. Structure of DNP, an N-nitroso compound. B. 6-10B cells were treated with the indicated concentration of DNP for 24 h. C. 6-10B cells were treated with 80 μmol/L DNP for the indicated time. CLU, MMP-9 and VEGF expressions in the DNP-treated cells were detected using Western-blotting (a). Three independent experiments were carried out, abundance ratio to GAPDH was counted, and data are represented as mean ±S.D. from three experiments (b). MMP-9 activity was measured using Fluorescent assay (c). DNP, N,N′-Dinitrosopiperazine; CLU, clusterin; MMP-9, matrix metalloproteinases 9; VEGF, vascular endothelial growth factor. * p

    Journal: Oncotarget

    Article Title: Clusterin induced by N,N′-Dinitrosopiperazine is involved in nasopharyngeal carcinoma metastasis

    doi: 10.18632/oncotarget.6750

    Figure Lengend Snippet: DNP induces expressions of CLU, MMP-9 and VEGF A. Structure of DNP, an N-nitroso compound. B. 6-10B cells were treated with the indicated concentration of DNP for 24 h. C. 6-10B cells were treated with 80 μmol/L DNP for the indicated time. CLU, MMP-9 and VEGF expressions in the DNP-treated cells were detected using Western-blotting (a). Three independent experiments were carried out, abundance ratio to GAPDH was counted, and data are represented as mean ±S.D. from three experiments (b). MMP-9 activity was measured using Fluorescent assay (c). DNP, N,N′-Dinitrosopiperazine; CLU, clusterin; MMP-9, matrix metalloproteinases 9; VEGF, vascular endothelial growth factor. * p

    Article Snippet: Enzymatic activity assay Activities of MMP-9 in the cells and NPC biopsy tissues were assayed using Cell or Tissue Active MMP-9 Fluorescent Assay Kit (Genmed Scientifics Inc. USA) following the manufacturer's instructions.

    Techniques: Concentration Assay, Western Blot, Activity Assay, Fluorescence

    DNP-induced NPC cell invasion and motility through CLU A. 6-10B cells were transiently transfected with siCLU or simock, and then treated with DNP. CLU, MMP-9 and VEGF expressions were detected in the transfected cells with or without DNP treatment using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). RNA transcriptions of CLU, MMP-9 and VEGF were analyzed using qRT-PCR (c). The motility and invasion of 6-10B-siCLU and 6-10B-simock were measured using Boden chamber invasion assay (d), and the invaded cells were counted (e). B. 6-10B cells were transfected with pcDNA3.1 (mock) or pcDNA3.1-CLU (CLU), and the stable cell lines 6-10B-mock and 6-10B-CLU were obtained using G418 selection. CLU, MMP-9 and VEGF expression were detected in the cells using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). The motility and invasion of 6-10B-mock and 6-10B-CLU were measured using Boden chamber invasion assay (c), the invaded cells were counted (d). Data are presented as means±SD from three independent experiments. Results were analyzed by one-way ANOVA with post hoc Dunnett's test. DNP, N,N′- Dinitrosopiperazine; CLU, clusterin; MMP-9, matrix metalloproteinases; VEGF, vascular endothelial growth factor. * p

    Journal: Oncotarget

    Article Title: Clusterin induced by N,N′-Dinitrosopiperazine is involved in nasopharyngeal carcinoma metastasis

    doi: 10.18632/oncotarget.6750

    Figure Lengend Snippet: DNP-induced NPC cell invasion and motility through CLU A. 6-10B cells were transiently transfected with siCLU or simock, and then treated with DNP. CLU, MMP-9 and VEGF expressions were detected in the transfected cells with or without DNP treatment using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). RNA transcriptions of CLU, MMP-9 and VEGF were analyzed using qRT-PCR (c). The motility and invasion of 6-10B-siCLU and 6-10B-simock were measured using Boden chamber invasion assay (d), and the invaded cells were counted (e). B. 6-10B cells were transfected with pcDNA3.1 (mock) or pcDNA3.1-CLU (CLU), and the stable cell lines 6-10B-mock and 6-10B-CLU were obtained using G418 selection. CLU, MMP-9 and VEGF expression were detected in the cells using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). The motility and invasion of 6-10B-mock and 6-10B-CLU were measured using Boden chamber invasion assay (c), the invaded cells were counted (d). Data are presented as means±SD from three independent experiments. Results were analyzed by one-way ANOVA with post hoc Dunnett's test. DNP, N,N′- Dinitrosopiperazine; CLU, clusterin; MMP-9, matrix metalloproteinases; VEGF, vascular endothelial growth factor. * p

    Article Snippet: Enzymatic activity assay Activities of MMP-9 in the cells and NPC biopsy tissues were assayed using Cell or Tissue Active MMP-9 Fluorescent Assay Kit (Genmed Scientifics Inc. USA) following the manufacturer's instructions.

    Techniques: Transfection, Western Blot, Quantitative RT-PCR, Invasion Assay, Stable Transfection, Selection, Expressing

    CLU knockdown reduces the invasion and metastasis of 5-8F cell A. 5-8F cells were transiently transfected with siCLU or simock. MMP-9 and VEGF expression were detected in these cells using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). Motility and invasion of the transfected cells were measured using Boden chamber invasion assay (c), and the invaded cells were counted (d). mRNA transcriptions of CLU, MMP-9 and VRGF were detected in the transfected cells using qRT-PCR (e). B. 5-8F cells were transfected with pSR-GFP/Neo -CLU-shRNA (shCLU) or pSR-GFP/Neo-NC -shRNA (shmock). 5-8F-shCLU and 5-8F-shmock stable-expressed cell lines were obtained using G418 selection. CLU, MMP-9 and VEGF expression were detected in the stable cells using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). The motility and invasion of 5-8F-shCLU and 5-8F-shmock were measured using Boden chamber invasion assay (c), and the invaded cells were counted (d). Data were from three independent experiments, expressed as means±SD. * p

    Journal: Oncotarget

    Article Title: Clusterin induced by N,N′-Dinitrosopiperazine is involved in nasopharyngeal carcinoma metastasis

    doi: 10.18632/oncotarget.6750

    Figure Lengend Snippet: CLU knockdown reduces the invasion and metastasis of 5-8F cell A. 5-8F cells were transiently transfected with siCLU or simock. MMP-9 and VEGF expression were detected in these cells using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). Motility and invasion of the transfected cells were measured using Boden chamber invasion assay (c), and the invaded cells were counted (d). mRNA transcriptions of CLU, MMP-9 and VRGF were detected in the transfected cells using qRT-PCR (e). B. 5-8F cells were transfected with pSR-GFP/Neo -CLU-shRNA (shCLU) or pSR-GFP/Neo-NC -shRNA (shmock). 5-8F-shCLU and 5-8F-shmock stable-expressed cell lines were obtained using G418 selection. CLU, MMP-9 and VEGF expression were detected in the stable cells using Western-blotting (a), and the abundance ratios to GAPDH were calculated (b). The motility and invasion of 5-8F-shCLU and 5-8F-shmock were measured using Boden chamber invasion assay (c), and the invaded cells were counted (d). Data were from three independent experiments, expressed as means±SD. * p

    Article Snippet: Enzymatic activity assay Activities of MMP-9 in the cells and NPC biopsy tissues were assayed using Cell or Tissue Active MMP-9 Fluorescent Assay Kit (Genmed Scientifics Inc. USA) following the manufacturer's instructions.

    Techniques: Transfection, Expressing, Western Blot, Invasion Assay, Quantitative RT-PCR, shRNA, Selection

    The immunohistochemical staining of CLU, MMP-9 and VEGF in NPC tissues A. Representative images of CLU, MMP-9 and VEGF protein expression in paraffin-embedded tissue from patients with NPC. CLU, clusterin; MMP-9, matrix metalloproteinases 9; VEGF, vascular endothelial growth factor; HE, hematoxylin and eosin. Original magnification, ×400. Scale bar, 5μm. B. Graphical illustration of statistical CLU, MMP-9 and VEGF distribution in NPC tissues. T, original tumor size and nearby tissue invasion; N, lymph node metastasis; M, distant metastasis. * p

    Journal: Oncotarget

    Article Title: Clusterin induced by N,N′-Dinitrosopiperazine is involved in nasopharyngeal carcinoma metastasis

    doi: 10.18632/oncotarget.6750

    Figure Lengend Snippet: The immunohistochemical staining of CLU, MMP-9 and VEGF in NPC tissues A. Representative images of CLU, MMP-9 and VEGF protein expression in paraffin-embedded tissue from patients with NPC. CLU, clusterin; MMP-9, matrix metalloproteinases 9; VEGF, vascular endothelial growth factor; HE, hematoxylin and eosin. Original magnification, ×400. Scale bar, 5μm. B. Graphical illustration of statistical CLU, MMP-9 and VEGF distribution in NPC tissues. T, original tumor size and nearby tissue invasion; N, lymph node metastasis; M, distant metastasis. * p

    Article Snippet: Enzymatic activity assay Activities of MMP-9 in the cells and NPC biopsy tissues were assayed using Cell or Tissue Active MMP-9 Fluorescent Assay Kit (Genmed Scientifics Inc. USA) following the manufacturer's instructions.

    Techniques: Immunohistochemistry, Staining, Expressing

    Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated activated primary peripheral monocytes. (a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) using anti-MMP-9 antibodies. The figure shows one representative analysis out of three. WIN inhibited MMP-9 secretion and induced an intracellular accumulation of 92 kDa MMP-9. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 and 4 µM WIN, a concentration-dependent reduction of MMP-9-activity was observed. Data are shown as means +/− SD n = 3. **p

    Journal: PLoS ONE

    Article Title: Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

    doi: 10.1371/journal.pone.0048272

    Figure Lengend Snippet: Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated activated primary peripheral monocytes. (a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) using anti-MMP-9 antibodies. The figure shows one representative analysis out of three. WIN inhibited MMP-9 secretion and induced an intracellular accumulation of 92 kDa MMP-9. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 and 4 µM WIN, a concentration-dependent reduction of MMP-9-activity was observed. Data are shown as means +/− SD n = 3. **p

    Article Snippet: MMP-9 Activity ELISA of Conditioned Medium MMP-9 activity in conditioned medium of U937-macrophages, primary human macrophages and osteoclastic cells was quantified by the ELISA Kit Fluorokine E human Active MMP-9 (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Inhibition, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Treatment with WIN reduced MMP-9 protein in bronchoalveolar lavage fluid (BALF) of mice with smoke-induced lung inflammation. Mice were exposed to air, cigarette smoke (smoke), or cigarette smoke plus i.p. treatment with 5 mg/kg/d WIN (smoke + WIN). (a) MMP-9 protein was measured by ELISA in BALF. Cigarette smoke-exposure enhances the MMP-9-content of BALF. I.p application of WIN during cigarette smoke-exposure reduced MMP-9 in BALF. (b) Number of white blood cells (WBCs) in BALF measured by haemocytometry. Cigarette smoke-exposure enhanced the content of WBCs in BALF significantly. I.p. application of WIN during cigarette smoke-exposure did not alter the number of WBCs. (c) Ratio of MMP-9/10 5 WBCs. The amount of MMP-9 per WBC decreased upon i.p. application of WIN significantly. Data are shown as means +/− SD, n = 7 (air) n = 8, (smoke), n = 9 (smoke+WIN). *p

    Journal: PLoS ONE

    Article Title: Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

    doi: 10.1371/journal.pone.0048272

    Figure Lengend Snippet: Treatment with WIN reduced MMP-9 protein in bronchoalveolar lavage fluid (BALF) of mice with smoke-induced lung inflammation. Mice were exposed to air, cigarette smoke (smoke), or cigarette smoke plus i.p. treatment with 5 mg/kg/d WIN (smoke + WIN). (a) MMP-9 protein was measured by ELISA in BALF. Cigarette smoke-exposure enhances the MMP-9-content of BALF. I.p application of WIN during cigarette smoke-exposure reduced MMP-9 in BALF. (b) Number of white blood cells (WBCs) in BALF measured by haemocytometry. Cigarette smoke-exposure enhanced the content of WBCs in BALF significantly. I.p. application of WIN during cigarette smoke-exposure did not alter the number of WBCs. (c) Ratio of MMP-9/10 5 WBCs. The amount of MMP-9 per WBC decreased upon i.p. application of WIN significantly. Data are shown as means +/− SD, n = 7 (air) n = 8, (smoke), n = 9 (smoke+WIN). *p

    Article Snippet: MMP-9 Activity ELISA of Conditioned Medium MMP-9 activity in conditioned medium of U937-macrophages, primary human macrophages and osteoclastic cells was quantified by the ELISA Kit Fluorokine E human Active MMP-9 (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    WIN reduced bone resorption and MMP-9-activity in a capsaicin sensitive manner. (a) Measurement of resorption activity of osteoclasts using crosslaps-ELISA of conditioned medium. Treatment with WIN (4 µM) reduced the osteolytic activity compared to control cells (vehicle treated). Additional treatment with capsaicin (CIC) antagonized this decrease. Data are shown as means +/− SD, n = 5. (b) MMP-9-activity-ELISA of conditioned medium of osteoclasts. Treatment with WIN (4 µM) decreased MMP-9-activity significantly compared to control cells (vehicle treated) and this decrease was antagonized by parallel treatment with CIC. Data are shown as mean +/− SD, n = 5. *p

    Journal: PLoS ONE

    Article Title: Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

    doi: 10.1371/journal.pone.0048272

    Figure Lengend Snippet: WIN reduced bone resorption and MMP-9-activity in a capsaicin sensitive manner. (a) Measurement of resorption activity of osteoclasts using crosslaps-ELISA of conditioned medium. Treatment with WIN (4 µM) reduced the osteolytic activity compared to control cells (vehicle treated). Additional treatment with capsaicin (CIC) antagonized this decrease. Data are shown as means +/− SD, n = 5. (b) MMP-9-activity-ELISA of conditioned medium of osteoclasts. Treatment with WIN (4 µM) decreased MMP-9-activity significantly compared to control cells (vehicle treated) and this decrease was antagonized by parallel treatment with CIC. Data are shown as mean +/− SD, n = 5. *p

    Article Snippet: MMP-9 Activity ELISA of Conditioned Medium MMP-9 activity in conditioned medium of U937-macrophages, primary human macrophages and osteoclastic cells was quantified by the ELISA Kit Fluorokine E human Active MMP-9 (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

    Treatment with WIN reduced secretion and activity of MMP-9 in macrophageal differentiated U937 cells. (a) Western blot analysis of conditioned medium using antibodies against MMP-9 and MMP-12. WIN-treatment resulted in a significant decrease of secreted MMP-9, whereas MMP-12 secretion was not affected. Control cells were treated with vehicle. The figure shows one representative analysis out of three. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 µM WIN a reduction of MMP-9 activity was observed, after treatment with 4 µM WIN the reduction was even stronger. Control cells were treated with vehicle. Data are shown as means +/− SD, n = 3. *p

    Journal: PLoS ONE

    Article Title: Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

    doi: 10.1371/journal.pone.0048272

    Figure Lengend Snippet: Treatment with WIN reduced secretion and activity of MMP-9 in macrophageal differentiated U937 cells. (a) Western blot analysis of conditioned medium using antibodies against MMP-9 and MMP-12. WIN-treatment resulted in a significant decrease of secreted MMP-9, whereas MMP-12 secretion was not affected. Control cells were treated with vehicle. The figure shows one representative analysis out of three. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 µM WIN a reduction of MMP-9 activity was observed, after treatment with 4 µM WIN the reduction was even stronger. Control cells were treated with vehicle. Data are shown as means +/− SD, n = 3. *p

    Article Snippet: MMP-9 Activity ELISA of Conditioned Medium MMP-9 activity in conditioned medium of U937-macrophages, primary human macrophages and osteoclastic cells was quantified by the ELISA Kit Fluorokine E human Active MMP-9 (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

    TRPV1 was involved in inhibition of secretion and intracellular accumulation of MMP-9 upon WIN-treatment. (a,b) Western blot analyses of U937-macrophage cell lysates (MMP-9 cellular) using MMP-9 antibody. (a) Treatment with the TRPV1 antagonist capsazepine (CZP) enhanced the WIN-induced size shift of MMP-9 from 85 to 92 kDa when given parallel to WIN and mimicked this effect when administered separately. The figure shows one representative analysis out of three. (b) Treatment with the TRPV1 agonist capsaicin (CIC) antagonized the WIN-induced size shift while it exhibited no effect when given alone. The figure shows one representative analysis out of three. (c) MMP-9 activity–ELISA of conditioned medium. The WIN-induced decrease of MMP-9 activity was intensified by CZP (10 µM), and antagonized by CIC (10µM). Data are shown as means +/− SD, n = 3. **p

    Journal: PLoS ONE

    Article Title: Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

    doi: 10.1371/journal.pone.0048272

    Figure Lengend Snippet: TRPV1 was involved in inhibition of secretion and intracellular accumulation of MMP-9 upon WIN-treatment. (a,b) Western blot analyses of U937-macrophage cell lysates (MMP-9 cellular) using MMP-9 antibody. (a) Treatment with the TRPV1 antagonist capsazepine (CZP) enhanced the WIN-induced size shift of MMP-9 from 85 to 92 kDa when given parallel to WIN and mimicked this effect when administered separately. The figure shows one representative analysis out of three. (b) Treatment with the TRPV1 agonist capsaicin (CIC) antagonized the WIN-induced size shift while it exhibited no effect when given alone. The figure shows one representative analysis out of three. (c) MMP-9 activity–ELISA of conditioned medium. The WIN-induced decrease of MMP-9 activity was intensified by CZP (10 µM), and antagonized by CIC (10µM). Data are shown as means +/− SD, n = 3. **p

    Article Snippet: MMP-9 Activity ELISA of Conditioned Medium MMP-9 activity in conditioned medium of U937-macrophages, primary human macrophages and osteoclastic cells was quantified by the ELISA Kit Fluorokine E human Active MMP-9 (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Inhibition, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated osteoclasts, but not in microglia. (a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted). using anti-MMP-9-antibody and MMP-9-activity ELISA of conditioned medium (bar chart) from osteoclasts. Upon WIN treatment (4 µM), the amount of intracellular 92 kDa-MMP-9 was enhanced, while the amount of secreted MMP-9 and the activity of MMP-9 in the conditioned medium was decreased. (b) Western blot analysis of cell lysates (MMP-9 cellular) using anti-MMP-9-antibody and MMP-9 ELISA of conditioned medium (bar chart) from primary microglia. Size and amount of intracellular MMP-9 were not changed after WIN-treatment (4 µM). The amount of MMP-9 in the conditioned medium increased insignificantly. PC = positive control (U937 macrophages). The figure shows one representative analysis out of three. Data are shown as means +/− SD n = 3. *p

    Journal: PLoS ONE

    Article Title: Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

    doi: 10.1371/journal.pone.0048272

    Figure Lengend Snippet: Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated osteoclasts, but not in microglia. (a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted). using anti-MMP-9-antibody and MMP-9-activity ELISA of conditioned medium (bar chart) from osteoclasts. Upon WIN treatment (4 µM), the amount of intracellular 92 kDa-MMP-9 was enhanced, while the amount of secreted MMP-9 and the activity of MMP-9 in the conditioned medium was decreased. (b) Western blot analysis of cell lysates (MMP-9 cellular) using anti-MMP-9-antibody and MMP-9 ELISA of conditioned medium (bar chart) from primary microglia. Size and amount of intracellular MMP-9 were not changed after WIN-treatment (4 µM). The amount of MMP-9 in the conditioned medium increased insignificantly. PC = positive control (U937 macrophages). The figure shows one representative analysis out of three. Data are shown as means +/− SD n = 3. *p

    Article Snippet: MMP-9 Activity ELISA of Conditioned Medium MMP-9 activity in conditioned medium of U937-macrophages, primary human macrophages and osteoclastic cells was quantified by the ELISA Kit Fluorokine E human Active MMP-9 (R & D Systems) according to the manufacturer’s instructions.

    Techniques: Inhibition, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control

    Endogenous βig-h3 cleavage by MMP-9 in glioma cells. A and B, βig-h3 and MMP-9 in MMP-9-transfected U87MG cells. A, βig-h3 and MMP-9 expression in the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells were examined by immunoblotting. B, MMP-9 activity was detected by gelatin zymography. M is a marker for active MMP-9 and pro-MMP-2 ( n = 3). C, co-immunoprecipitation of MMP-9 with βig-h3 from the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells. After the lysates from the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells were immunoprecipitated ( IP ) with anti-βig-h3 (+) or control rabbit IgG (−), they were subjected to immunoblotting ( IB ) using mouse antibody for Myc ( MMP-9 ) ( n = 4). D, MMP-9 overexpressed in cells alters cell invasion. Cell invasion in vehicle-transfected, MMP-9-transfected, or IL-1β (10 ng/ml)-treated U87MG cells was examined on Matrigels. βig-h3 and MMP-9 proteins from IL-1β-treated human glioma U87MG cells were examined by immunoblotting and zymography. The control represents untreated samples ( n = 3). E, βig-h3 knockdown alters cell invasion. Cell invasion in control siRNA or βig-h3 siRNA-transfected U87MG cells was examined on Matrigels. The βig-h3 and MMP-9 from these cells were examined by immunoblotting and zymography. Control represents control siRNA. (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Matrix Metalloproteinase 9 (MMP-9)-dependent Processing of ?ig-h3 Protein Regulates Cell Migration, Invasion, and Adhesion *

    doi: 10.1074/jbc.M112.357863

    Figure Lengend Snippet: Endogenous βig-h3 cleavage by MMP-9 in glioma cells. A and B, βig-h3 and MMP-9 in MMP-9-transfected U87MG cells. A, βig-h3 and MMP-9 expression in the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells were examined by immunoblotting. B, MMP-9 activity was detected by gelatin zymography. M is a marker for active MMP-9 and pro-MMP-2 ( n = 3). C, co-immunoprecipitation of MMP-9 with βig-h3 from the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells. After the lysates from the vehicle ( vehicle/U87MG )-transfected or MMP-9 ( MMP-9/U87MG )-transfected U87MG cells were immunoprecipitated ( IP ) with anti-βig-h3 (+) or control rabbit IgG (−), they were subjected to immunoblotting ( IB ) using mouse antibody for Myc ( MMP-9 ) ( n = 4). D, MMP-9 overexpressed in cells alters cell invasion. Cell invasion in vehicle-transfected, MMP-9-transfected, or IL-1β (10 ng/ml)-treated U87MG cells was examined on Matrigels. βig-h3 and MMP-9 proteins from IL-1β-treated human glioma U87MG cells were examined by immunoblotting and zymography. The control represents untreated samples ( n = 3). E, βig-h3 knockdown alters cell invasion. Cell invasion in control siRNA or βig-h3 siRNA-transfected U87MG cells was examined on Matrigels. The βig-h3 and MMP-9 from these cells were examined by immunoblotting and zymography. Control represents control siRNA. (*, p

    Article Snippet: Commercially available recombinant human active MMP-9 catalytic subunit (ProSpec), recombinant human full-length pro-MMP-9 monomer (Calbiochem), recombinant human βig-h3 (Sino Biological), recombinant human βig-h3 fragment (ProSpec, βig-h3–3rd), βig-h3-mutant from βig-h3-overexpressing 293F cells, and pro-MMP-9 from MMP-9-overexpressing 293F cells were used.

    Techniques: Transfection, Expressing, Activity Assay, Zymography, Marker, Immunoprecipitation

    Marimastat blocks forward neuroblast migration. ( A – C ) Focal administration of the dual MMP-2/MMP-9 inhibitor Marimastat (1 µ M/0.1 µL) (Tocris) increased the diameter of the proximal RMS ( B 2 ) but decreased the diameter of the distal

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration

    doi: 10.1073/pnas.1700662114

    Figure Lengend Snippet: Marimastat blocks forward neuroblast migration. ( A – C ) Focal administration of the dual MMP-2/MMP-9 inhibitor Marimastat (1 µ M/0.1 µL) (Tocris) increased the diameter of the proximal RMS ( B 2 ) but decreased the diameter of the distal

    Article Snippet: Rats ( n = 6) received Marimastat (1 µM/0.1 µL) (Tocris), a potent broad-spectrum MMP inhibitor with anti–MMP-2/MMP-9 activity , or DMSO (0.1/µL) (Sigma) in the distal limb of the RMS (at coordinates 1.2 mm lateral and 3.0 mm rostral from bregma, 6.0 mm ventral from the dural surface).

    Techniques: Migration

    Concentrations of proteins in peripheral blood lysates in control and sarcoidosis groups for: ( A ) MMP-2; ( B ) MMP-7; ( C ) MMP-9; ( D ) TIMP-2. Data shown as median (horizontal line) and interquartile range (boxes). Whiskers show minimum and maximum values.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: The selected genetic polymorphisms of metalloproteinases MMP2, 7, 9 and MMP inhibitor TIMP2 in sarcoidosis

    doi: 10.12659/MSM.881987

    Figure Lengend Snippet: Concentrations of proteins in peripheral blood lysates in control and sarcoidosis groups for: ( A ) MMP-2; ( B ) MMP-7; ( C ) MMP-9; ( D ) TIMP-2. Data shown as median (horizontal line) and interquartile range (boxes). Whiskers show minimum and maximum values.

    Article Snippet: MMP-2/MMP-9, human zymography standards (Millipore, Billerica, MA, US), were simultaneously loaded onto the gel.

    Techniques:

    Gelatin substrate zymography reveals that MMP-9 activity was increased > 4-fold in MCT sl/sl lungs compared with MCT +/+ lungs, whereas MMP-2 activity was not different between groups. Note that MMP-2 and MMP-9 are detected as both a higher-molecular-weight

    Journal:

    Article Title: Development of Occlusive Neointimal Lesions in Distal Pulmonary Arteries of Endothelin B Receptor-Deficient Rats: A New Model of Severe Pulmonary Arterial Hypertension

    doi: 10.1161/CIRCULATIONAHA.104.491456

    Figure Lengend Snippet: Gelatin substrate zymography reveals that MMP-9 activity was increased > 4-fold in MCT sl/sl lungs compared with MCT +/+ lungs, whereas MMP-2 activity was not different between groups. Note that MMP-2 and MMP-9 are detected as both a higher-molecular-weight

    Article Snippet: To determine whether matrix metalloproteinases (MMPs) in lung tissue are equivalent in molecular size and activity to MMP-9 and MMP-2, a purified MMP-9/MMP-2 standard (Sigma) was also separated on substrate gels.

    Techniques: Zymography, Activity Assay, Molecular Weight

    Mesenchymal leader cells in the 2D ex vivo injury culture model are enriched for molecules involved in invasion, CD44, MMP-2, and MMP-9. (A) Model of the different regions of the ex vivo mock cataract surgery explant that are separated by microdissection for this study from both top and side views. Cells found in the points of the star-shaped culture that contain the region of the lens that is occupied by epithelial cells in vivo are referred to as the original attachment zone (OAZ, yellow). In response to injury, cells move onto the denuded basement membrane (BM) into the central migration zone (CMZ, purple). (B–D) To examine whether there are migration-specific changes in expression of molecules associated with invasion after wounding, ex vivo injury explants were microdissected into OAZ and CMZ regions and extracted on days 1–3 for Western blot analysis for CD44, MMP-9, MMP-2, and GAPDH (loading control). Graphs depicting Western blot results from four independent studies show the relative expression of CD44, MMP-9, and MMP-2 to GAPDH. CD44, MMP-9, and MMP-2 were predominately expressed in the CMZ region. These molecules decreased in expression as wound healing progressed on day 2 and on day 3, when wound healing is typically completed. MMP-2 and MMP-9 were detected predominantly in their active forms (bottom arrows) compared with their inactive proenzymatic form (top arrows). For both MMP-2 and MMP-9, the active form (lower band) was quantified and is represented on the graphs as a ratio to GAPDH. Immunoblots were quantified using Kodak 1D software, and data were normalized to results at day 1 in the CMZ zone. Values for each graph and relative densities are plotted ±SEM. Representative Western blots are shown below each graph. (E–M) Ex vivo–wounded cultures were fixed and immunostained for vimentin (F, I, L; green) and colabeled for CD44 (E, G; red), MMP-9 (H, J; red), or MMP-2 (K, M; red). Vimentin-rich mesenchymal cells at the leading edge were enriched for MMP-2, MMP-9, and CD44. CD44 localized at cell–cell interfaces and to the tips of these cells at the leading edge (E–G, arrow). MMP-9 strongly localized to vimentin-rich cells, sometimes colocalized with vimentin (H–J, arrow) and also to regions in the area to be repaired just beyond the cells (H, J, arrowhead). Bar, 10 μm. (N) The role of MMP-2/9 function on wound healing in the 2D ex vivo injury cultures was evaluated using inhibitors to MMP-2, MMP-9, or MMP-2/9. Phase images were taken on days 0–3, from which the open wound area was calculated in at least six capsules and quantified using NIS Elements analysis software. Open wound area was plotted on the graphs ±SEM. Inhibition of MMP-2 and/or MMP-9 function resulted in only a slight delay in wound closure.

    Journal: Molecular Biology of the Cell

    Article Title: Cells activated for wound repair have the potential to direct collective invasion of an epithelium

    doi: 10.1091/mbc.E15-09-0615

    Figure Lengend Snippet: Mesenchymal leader cells in the 2D ex vivo injury culture model are enriched for molecules involved in invasion, CD44, MMP-2, and MMP-9. (A) Model of the different regions of the ex vivo mock cataract surgery explant that are separated by microdissection for this study from both top and side views. Cells found in the points of the star-shaped culture that contain the region of the lens that is occupied by epithelial cells in vivo are referred to as the original attachment zone (OAZ, yellow). In response to injury, cells move onto the denuded basement membrane (BM) into the central migration zone (CMZ, purple). (B–D) To examine whether there are migration-specific changes in expression of molecules associated with invasion after wounding, ex vivo injury explants were microdissected into OAZ and CMZ regions and extracted on days 1–3 for Western blot analysis for CD44, MMP-9, MMP-2, and GAPDH (loading control). Graphs depicting Western blot results from four independent studies show the relative expression of CD44, MMP-9, and MMP-2 to GAPDH. CD44, MMP-9, and MMP-2 were predominately expressed in the CMZ region. These molecules decreased in expression as wound healing progressed on day 2 and on day 3, when wound healing is typically completed. MMP-2 and MMP-9 were detected predominantly in their active forms (bottom arrows) compared with their inactive proenzymatic form (top arrows). For both MMP-2 and MMP-9, the active form (lower band) was quantified and is represented on the graphs as a ratio to GAPDH. Immunoblots were quantified using Kodak 1D software, and data were normalized to results at day 1 in the CMZ zone. Values for each graph and relative densities are plotted ±SEM. Representative Western blots are shown below each graph. (E–M) Ex vivo–wounded cultures were fixed and immunostained for vimentin (F, I, L; green) and colabeled for CD44 (E, G; red), MMP-9 (H, J; red), or MMP-2 (K, M; red). Vimentin-rich mesenchymal cells at the leading edge were enriched for MMP-2, MMP-9, and CD44. CD44 localized at cell–cell interfaces and to the tips of these cells at the leading edge (E–G, arrow). MMP-9 strongly localized to vimentin-rich cells, sometimes colocalized with vimentin (H–J, arrow) and also to regions in the area to be repaired just beyond the cells (H, J, arrowhead). Bar, 10 μm. (N) The role of MMP-2/9 function on wound healing in the 2D ex vivo injury cultures was evaluated using inhibitors to MMP-2, MMP-9, or MMP-2/9. Phase images were taken on days 0–3, from which the open wound area was calculated in at least six capsules and quantified using NIS Elements analysis software. Open wound area was plotted on the graphs ±SEM. Inhibition of MMP-2 and/or MMP-9 function resulted in only a slight delay in wound closure.

    Article Snippet: To inhibit MMP-2 and/or MMP-9 function, ex vivo explants were grown in the presence of inhibitors to MMP-2 (20 μM; Millipore, Billerica, MA), MMP-9 (20 μM; Millipore), or MMP-2/9 (25 μM; Millipore).

    Techniques: Ex Vivo, Laser Capture Microdissection, In Vivo, Migration, Expressing, Western Blot, Software, Inhibition

    Functional properties of invading cells in response to injury. (A–G) The 3D Matrigel invasion assay of ex vivo–wounded explants at (A–D) day 6 in culture and (E–G) day 2 in culture. (A) A 3D reconstruction of a confocal Z -stack of cords of cells in the injury invasion assay labeled for vimentin (green) and F-actin (red), emphasizing the extension of vimentin-rich cells into the Matrigel matrix in leader-cell positions. Bar, 30 μm. (B, C) Cords of invading cells in the Matrigel invasion assay were immunolabeled for cortactin (red) and vimentin (green) and viewed by confocal microscopy. This projection image of a confocal Z -stack shows that the processes extended by the vimentin-rich mesenchymal cells into the Matrigel also expressed cortactin (red). Bar, 5 μm. (D) Labeling for MMP-9 (green) in cells at the leading edges of the cords extended into Matrigel and was distributed in discrete vesicular structures in the cells (F-actin; red) and also in a punctate pattern just beyond the cells in the Matrigel matrix, where it can act to degrade the matrix for invasion. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (E–G) Vimentin-rich cells at the invading front (green) also express αSMA (red), often with a punctate distribution (arrowhead). Labeling for F-actin (purple) was mostly absent from the areas with a strong distribution of αSMA puncta, suggesting that this actin population was nonfilamentous. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (H) The individual roles of MMP-2/9 and vimentin in invasion after wounding were evaluated using an inhibitor approach in the Matrigel-Transwell chemoinvasion assay. This study included the MMP-2 inhibitor MMP-2 i , the MMP-9 inhibitor MMP-9 i , the combined MMP-2/9 inhibitor MMP-2/9 i , and the vimentin inhibitor WFA. Invaded cells from at least 10 capsules were counted and quantified using MetaMorph or NIS Elements image analysis software. Relative numbers of invaded cells were plotted on graphs ±SEM. MMP-2 and MMP-9 function were required for invasion of cells in the Matrigel-Transwell assay. MMP-2 inhibited invasion by 99%, MMP-9 by 96%, and MMP2/9 by 84% in the Matrigel-Transwell assays. Inhibiting vimentin function with WFA suppressed invasion in the Matrigel-Transwell assay by 98%.

    Journal: Molecular Biology of the Cell

    Article Title: Cells activated for wound repair have the potential to direct collective invasion of an epithelium

    doi: 10.1091/mbc.E15-09-0615

    Figure Lengend Snippet: Functional properties of invading cells in response to injury. (A–G) The 3D Matrigel invasion assay of ex vivo–wounded explants at (A–D) day 6 in culture and (E–G) day 2 in culture. (A) A 3D reconstruction of a confocal Z -stack of cords of cells in the injury invasion assay labeled for vimentin (green) and F-actin (red), emphasizing the extension of vimentin-rich cells into the Matrigel matrix in leader-cell positions. Bar, 30 μm. (B, C) Cords of invading cells in the Matrigel invasion assay were immunolabeled for cortactin (red) and vimentin (green) and viewed by confocal microscopy. This projection image of a confocal Z -stack shows that the processes extended by the vimentin-rich mesenchymal cells into the Matrigel also expressed cortactin (red). Bar, 5 μm. (D) Labeling for MMP-9 (green) in cells at the leading edges of the cords extended into Matrigel and was distributed in discrete vesicular structures in the cells (F-actin; red) and also in a punctate pattern just beyond the cells in the Matrigel matrix, where it can act to degrade the matrix for invasion. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (E–G) Vimentin-rich cells at the invading front (green) also express αSMA (red), often with a punctate distribution (arrowhead). Labeling for F-actin (purple) was mostly absent from the areas with a strong distribution of αSMA puncta, suggesting that this actin population was nonfilamentous. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (H) The individual roles of MMP-2/9 and vimentin in invasion after wounding were evaluated using an inhibitor approach in the Matrigel-Transwell chemoinvasion assay. This study included the MMP-2 inhibitor MMP-2 i , the MMP-9 inhibitor MMP-9 i , the combined MMP-2/9 inhibitor MMP-2/9 i , and the vimentin inhibitor WFA. Invaded cells from at least 10 capsules were counted and quantified using MetaMorph or NIS Elements image analysis software. Relative numbers of invaded cells were plotted on graphs ±SEM. MMP-2 and MMP-9 function were required for invasion of cells in the Matrigel-Transwell assay. MMP-2 inhibited invasion by 99%, MMP-9 by 96%, and MMP2/9 by 84% in the Matrigel-Transwell assays. Inhibiting vimentin function with WFA suppressed invasion in the Matrigel-Transwell assay by 98%.

    Article Snippet: To inhibit MMP-2 and/or MMP-9 function, ex vivo explants were grown in the presence of inhibitors to MMP-2 (20 μM; Millipore, Billerica, MA), MMP-9 (20 μM; Millipore), or MMP-2/9 (25 μM; Millipore).

    Techniques: Functional Assay, Invasion Assay, Ex Vivo, Labeling, Immunolabeling, Confocal Microscopy, Activated Clotting Time Assay, Software, Transwell Assay

    FAK and activated Q61L Rac synergize to promote JNK activation, increased MMP-9 expression, and an invasive cell phenotype. (A) Flag tag blotting was used to visualize Q61L Rac expression in the indicated Ad- infected cells. (B) Matrigel invasion assays were performed with the indicated Mock or Ad-Q61L Rac-infected cells. Values are means ± SD of triplicates from two independent experiments. (C) Gelatin zymography was performed with conditioned media from the indicated Mock or Ad-Q61L Rac-infected cells. Migration of pro and active forms of MMP-9 and MMP-2 are shown. (D) Conditioned media from FAK −/− v-Src or DA2 v-Src cells was concentrated, and MMP-9 or MMP-2 secretion was evaluated by blotting. (E) Semiquantitative RT-PCR was performed using primers to MMP-9 and to β-actin and showed that MMP-9 mRNA levels were 6.5-fold higher in DA2 v-Src compared with FAK −/− v-Src cells. (F) Matrigel invasion assays were performed with DA2 v-Src cells with the addition of recombinant TIMP-1 at the indicated concentration in the top chamber. Values are means ± SD of triplicates from two independent experiments. (G) JNK IP/IVK assays performed with lysates from FAK −/− cells or Ad-Q61L Rac-infected FAK −/− , FAK −/− v-Src, or DA2 v-Src cells. Values are means ± SD of duplicates from two independent experiments. Flag tag blotting of whole cell lysates was used to verify equivalent Q61L Rac expression.

    Journal: The Journal of Cell Biology

    Article Title: Differential regulation of cell motility and invasion by FAK

    doi: 10.1083/jcb.200212114

    Figure Lengend Snippet: FAK and activated Q61L Rac synergize to promote JNK activation, increased MMP-9 expression, and an invasive cell phenotype. (A) Flag tag blotting was used to visualize Q61L Rac expression in the indicated Ad- infected cells. (B) Matrigel invasion assays were performed with the indicated Mock or Ad-Q61L Rac-infected cells. Values are means ± SD of triplicates from two independent experiments. (C) Gelatin zymography was performed with conditioned media from the indicated Mock or Ad-Q61L Rac-infected cells. Migration of pro and active forms of MMP-9 and MMP-2 are shown. (D) Conditioned media from FAK −/− v-Src or DA2 v-Src cells was concentrated, and MMP-9 or MMP-2 secretion was evaluated by blotting. (E) Semiquantitative RT-PCR was performed using primers to MMP-9 and to β-actin and showed that MMP-9 mRNA levels were 6.5-fold higher in DA2 v-Src compared with FAK −/− v-Src cells. (F) Matrigel invasion assays were performed with DA2 v-Src cells with the addition of recombinant TIMP-1 at the indicated concentration in the top chamber. Values are means ± SD of triplicates from two independent experiments. (G) JNK IP/IVK assays performed with lysates from FAK −/− cells or Ad-Q61L Rac-infected FAK −/− , FAK −/− v-Src, or DA2 v-Src cells. Values are means ± SD of duplicates from two independent experiments. Flag tag blotting of whole cell lysates was used to verify equivalent Q61L Rac expression.

    Article Snippet: Analysis of soluble MMP-2 + MMP-9 activity was performed using the Chemicon Gelatinase Activity kit (ECM700) as per the manufacturer's instructions.

    Techniques: Activation Assay, Expressing, FLAG-tag, Infection, Zymography, Migration, Reverse Transcription Polymerase Chain Reaction, Recombinant, Concentration Assay

    Models of differential FAK signaling connections at focal contacts or lamellipodia/invadopodia. (A) Integrin-associated FAK and Src activation promotes increased p190RhoGAP tyrosine phosphorylation or ERK2/MAP kinase activation. FAK–Src modulation of p21 Rho activity in FAK −/− cells is associated with increased cell motility, focal contact turnover, and actin cytoskeletal rearrangements. (B) In either EGF-stimulated human adenocarcinoma cells ( Hauck et al., 2001b ) or serum-stimulated v-Src–transformed fibroblasts, FAK functions to coordinate a Src-p130Cas signaling complex localized to lamellipodia in two-dimensional cell culture or to invadopodia in 3-D invasion assays. Through direct Cas SH3-mediated and indirect Crk-mediated interactions with the Dock180 guanine-nucleotide exchange factor, the FAK–Src signaling complex promotes Rac and JNK activation with effects upon gene expression. Although PI 3-kinase is a direct target of both v-Src and Rac ( Liu et al., 1993 ; Bishop and Hall, 2000 ), FAK −/− v-Src cells exhibit defects in JNK but not Akt activation. Overexpression of JNK in FAK −/− v-Src cells coupled with serum stimulation promoted c-Jun Ser-63 phosphorylation, MMP-9 expression, MMP-2 activation, and rescued the FAK −/− v-Src cell invasion defects.

    Journal: The Journal of Cell Biology

    Article Title: Differential regulation of cell motility and invasion by FAK

    doi: 10.1083/jcb.200212114

    Figure Lengend Snippet: Models of differential FAK signaling connections at focal contacts or lamellipodia/invadopodia. (A) Integrin-associated FAK and Src activation promotes increased p190RhoGAP tyrosine phosphorylation or ERK2/MAP kinase activation. FAK–Src modulation of p21 Rho activity in FAK −/− cells is associated with increased cell motility, focal contact turnover, and actin cytoskeletal rearrangements. (B) In either EGF-stimulated human adenocarcinoma cells ( Hauck et al., 2001b ) or serum-stimulated v-Src–transformed fibroblasts, FAK functions to coordinate a Src-p130Cas signaling complex localized to lamellipodia in two-dimensional cell culture or to invadopodia in 3-D invasion assays. Through direct Cas SH3-mediated and indirect Crk-mediated interactions with the Dock180 guanine-nucleotide exchange factor, the FAK–Src signaling complex promotes Rac and JNK activation with effects upon gene expression. Although PI 3-kinase is a direct target of both v-Src and Rac ( Liu et al., 1993 ; Bishop and Hall, 2000 ), FAK −/− v-Src cells exhibit defects in JNK but not Akt activation. Overexpression of JNK in FAK −/− v-Src cells coupled with serum stimulation promoted c-Jun Ser-63 phosphorylation, MMP-9 expression, MMP-2 activation, and rescued the FAK −/− v-Src cell invasion defects.

    Article Snippet: Analysis of soluble MMP-2 + MMP-9 activity was performed using the Chemicon Gelatinase Activity kit (ECM700) as per the manufacturer's instructions.

    Techniques: Activation Assay, Activity Assay, Transformation Assay, Cell Culture, Expressing, Over Expression

    The results of aortic valve interstitial cell culture studies in which expression of matrix metalloproteinase-2 (MMP-2) is up-regulated by tenascin-C (TN-C). A: relative amounts of mRNA in sheep (SAVIC) and human aortic valve interstitial cells (HAVIC), evaluated by competitive RT-PCR. Cells were grown on a substrate of either bovine type I collagen or collagen with TN-C (15 μg/ml). A representative agarose gel (see Materials and Methods) is shown for SAVIC demonstrating greater amounts of MMP-2 RNA in the TN-C cultures. The top bands are derived from MMP-2 reverse transcriptase (RT) products, while the bottom bands are due to the MMP-2M13 competitors. The lanes from left to right represent PCRs with a series of dilutions of the MMP-2M13 competitor. The reduction in MMP-2M13 band intensity (bottom bands) occurs in conjunction with an increase in the upper bands (MMP-2 RT). The last lane is a 100 bp DNA ladder. Bars represent the mean ± SE of 3 independent measurements. TN-C significantly increased MMP-2 expression ( P

    Journal: The American Journal of Pathology

    Article Title: Matrix Metalloproteinase-2 Is Associated with Tenascin-C in Calcific Aortic Stenosis

    doi:

    Figure Lengend Snippet: The results of aortic valve interstitial cell culture studies in which expression of matrix metalloproteinase-2 (MMP-2) is up-regulated by tenascin-C (TN-C). A: relative amounts of mRNA in sheep (SAVIC) and human aortic valve interstitial cells (HAVIC), evaluated by competitive RT-PCR. Cells were grown on a substrate of either bovine type I collagen or collagen with TN-C (15 μg/ml). A representative agarose gel (see Materials and Methods) is shown for SAVIC demonstrating greater amounts of MMP-2 RNA in the TN-C cultures. The top bands are derived from MMP-2 reverse transcriptase (RT) products, while the bottom bands are due to the MMP-2M13 competitors. The lanes from left to right represent PCRs with a series of dilutions of the MMP-2M13 competitor. The reduction in MMP-2M13 band intensity (bottom bands) occurs in conjunction with an increase in the upper bands (MMP-2 RT). The last lane is a 100 bp DNA ladder. Bars represent the mean ± SE of 3 independent measurements. TN-C significantly increased MMP-2 expression ( P

    Article Snippet: Mouse monoclonal antibodies against human TN-C (Life Technologies) and MMP-2 (Oncogene Research Products, Cambridge, MA) were used.

    Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Derivative Assay

    MMP-2 activity was present in individual extracts of five explanted aortic valve cusps obtained at surgery for calcific aortic stenosis. Gel zymography shows MMP-2 (pro form only) in all cases ( lanes 2–6 ), and qualitatively lesser amounts MMP-9 in cases 1, 2, and 3 ( lanes 2 - 4 ) as compared to MMP standards ( lane 7 ). Lane 1 contains molecular weight standards.

    Journal: The American Journal of Pathology

    Article Title: Matrix Metalloproteinase-2 Is Associated with Tenascin-C in Calcific Aortic Stenosis

    doi:

    Figure Lengend Snippet: MMP-2 activity was present in individual extracts of five explanted aortic valve cusps obtained at surgery for calcific aortic stenosis. Gel zymography shows MMP-2 (pro form only) in all cases ( lanes 2–6 ), and qualitatively lesser amounts MMP-9 in cases 1, 2, and 3 ( lanes 2 - 4 ) as compared to MMP standards ( lane 7 ). Lane 1 contains molecular weight standards.

    Article Snippet: Mouse monoclonal antibodies against human TN-C (Life Technologies) and MMP-2 (Oncogene Research Products, Cambridge, MA) were used.

    Techniques: Activity Assay, Zymography, Molecular Weight

    LRP1 knockdown has no impact on the level of Aβ-degrading enzymes in C3H10T1/2 cell-derived pericytes The protein levels of Aβ-degrading enzymes (MMP2, MMP9, NEP, and IDE) in the pericytes treated with control or LRP1-siRNA were analyzed by Western blotting. Data are plotted as mean ± S.E.M. (n=3). n.s., not significant.

    Journal: Experimental neurology

    Article Title: Pericyte Implantation in the Brain Enhances Cerebral Blood Flow and Reduces Amyloid-β Pathology in Amyloid Model Mice

    doi: 10.1016/j.expneurol.2017.10.023

    Figure Lengend Snippet: LRP1 knockdown has no impact on the level of Aβ-degrading enzymes in C3H10T1/2 cell-derived pericytes The protein levels of Aβ-degrading enzymes (MMP2, MMP9, NEP, and IDE) in the pericytes treated with control or LRP1-siRNA were analyzed by Western blotting. Data are plotted as mean ± S.E.M. (n=3). n.s., not significant.

    Article Snippet: In addition, knockdown of LRP1 in C3H/10T1/2 cell-derived pericytes did not have substantial effects on Aβ degrading enzymes including NEP, IDE, MMP2, and MMP9 examined by Western blotting ( ).

    Techniques: Derivative Assay, Western Blot

    ( a ) Immunoblot analysis showing protein levels of CD133, as well as secreted MMP2 and MMP9, in different CRISPR-Cas9 melanoma cell lines and BAKP; β-Actin verified equal protein loading. Immunoblot analysis shows decreased levels of CD133 and secreted MMPs in CRISPR Cas-9 CD133 knockdown melanoma pooled clone T1 and T3, compared to BAKP and scrambled control cells. ( b ) Representative images of crystal violet stained invading cells after 48 hours in transwell invasion chambers; HT1080 and MCF-7 served as positive and negative controls, respectively. ( c ) Percent invasion of CRISPR Cas-9 CD133 knockdown cell lines (T1, T2, T3) compared to BAKP or scrambled (SC) control cells; *, **, *** represent p

    Journal: Cancers

    Article Title: CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis

    doi: 10.3390/cancers11101490

    Figure Lengend Snippet: ( a ) Immunoblot analysis showing protein levels of CD133, as well as secreted MMP2 and MMP9, in different CRISPR-Cas9 melanoma cell lines and BAKP; β-Actin verified equal protein loading. Immunoblot analysis shows decreased levels of CD133 and secreted MMPs in CRISPR Cas-9 CD133 knockdown melanoma pooled clone T1 and T3, compared to BAKP and scrambled control cells. ( b ) Representative images of crystal violet stained invading cells after 48 hours in transwell invasion chambers; HT1080 and MCF-7 served as positive and negative controls, respectively. ( c ) Percent invasion of CRISPR Cas-9 CD133 knockdown cell lines (T1, T2, T3) compared to BAKP or scrambled (SC) control cells; *, **, *** represent p

    Article Snippet: For secreted MMP2/MMP9, conditioned media was collected from the cells, concentrated by centrifugation at 14,000 g using 10 kDa Amicon® filters (MilliporeSigma, Burlington, MA, USA), and then separated by SDS-PAGE, and western transfer.

    Techniques: CRISPR, Staining

    Dox-inducible CD133 expression in BAK-P cells as verified by qPCR ( a ) and immunoblot analysis ( b ). ( c ) Dox-inducible CD133 expression significantly increases cell invasion in transwell assays. ( d ) Immunoblot analysis with antibodies to CD133, MMP9, MMP2, or β-Actin for loading controls, show increased levels of CD133 as well as MP2 and MMP9 secreted by Dox-induced cells. ( e ) Zebrafish assays reveal enhanced metastasis in Dox-induced cells. ( f , g left panels) Invasion assay results with other melanoma cell lines POT ( f ) and SK-MEL2 ( g ) CD133 CRISPR-cas9 SC versus T3 lines are consistent with results with BAK-P cells, showing that knockdown of CD133 ( f , g right panels) results in decreased cell invasion. *, **, *** represent p

    Journal: Cancers

    Article Title: CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis

    doi: 10.3390/cancers11101490

    Figure Lengend Snippet: Dox-inducible CD133 expression in BAK-P cells as verified by qPCR ( a ) and immunoblot analysis ( b ). ( c ) Dox-inducible CD133 expression significantly increases cell invasion in transwell assays. ( d ) Immunoblot analysis with antibodies to CD133, MMP9, MMP2, or β-Actin for loading controls, show increased levels of CD133 as well as MP2 and MMP9 secreted by Dox-induced cells. ( e ) Zebrafish assays reveal enhanced metastasis in Dox-induced cells. ( f , g left panels) Invasion assay results with other melanoma cell lines POT ( f ) and SK-MEL2 ( g ) CD133 CRISPR-cas9 SC versus T3 lines are consistent with results with BAK-P cells, showing that knockdown of CD133 ( f , g right panels) results in decreased cell invasion. *, **, *** represent p

    Article Snippet: For secreted MMP2/MMP9, conditioned media was collected from the cells, concentrated by centrifugation at 14,000 g using 10 kDa Amicon® filters (MilliporeSigma, Burlington, MA, USA), and then separated by SDS-PAGE, and western transfer.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Invasion Assay, CRISPR

    Recombinant MMP-9 (rMMP-9) induces lobar hemorrhage in brains of C57/BL6 mice (A) Representative images of mouse brains topically treated with or without rMMP-9. C57/BL6 mice at 3 months of age were prepared with a craniotomy followed by careful removal of the dura mater. Pre-activated recombinant MMP-9 (rMMP-9, 0.4 μg) or PBS (control) was topically applied to the surface of mouse brains. Forty eight hours after the treatment, the mice were sacrificed and mouse brains were removed to examine the lobar hemorrhage and imaged by a camera (upper panel) or a low magnification microscope (2.5X, Olympus) (lower panel). (B) Representative H E staining of brain sections from mice topically treated with or without rMMP-9. Mouse brains treated with rMMP-9 or PBS (control) were sectioned and processed for histologic examination by H E staining. Arrows show the vascular rupture in mouse brains treated with rMMP-9. Scale bar = 200 μm.

    Journal: Neurobiology of aging

    Article Title: Matrix Metalloproteinase 9 mediated intracerebral hemorrhage induced by Cerebral amyloid angiopathy

    doi: 10.1016/j.neurobiolaging.2015.07.016

    Figure Lengend Snippet: Recombinant MMP-9 (rMMP-9) induces lobar hemorrhage in brains of C57/BL6 mice (A) Representative images of mouse brains topically treated with or without rMMP-9. C57/BL6 mice at 3 months of age were prepared with a craniotomy followed by careful removal of the dura mater. Pre-activated recombinant MMP-9 (rMMP-9, 0.4 μg) or PBS (control) was topically applied to the surface of mouse brains. Forty eight hours after the treatment, the mice were sacrificed and mouse brains were removed to examine the lobar hemorrhage and imaged by a camera (upper panel) or a low magnification microscope (2.5X, Olympus) (lower panel). (B) Representative H E staining of brain sections from mice topically treated with or without rMMP-9. Mouse brains treated with rMMP-9 or PBS (control) were sectioned and processed for histologic examination by H E staining. Arrows show the vascular rupture in mouse brains treated with rMMP-9. Scale bar = 200 μm.

    Article Snippet: MMP-9 antibody (catalytic domain) and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Millipore; recombinant human MMP-9 (pre-activated) was purchased from Anaspec; monoclonal antibody against human fibrin (a′-fibrin) was purchased from American Diagnostic Inc.; The anti-fibrin antibody was labeled with DyLight 594 using a commercial kit (Thermo Scientific); Prussian blue staining kits were purchased from Polysciences, Inc.

    Techniques: Recombinant, Mouse Assay, Microscopy, Staining

    MMP-9 accumulates in CAA-affected vessels (A) Representative immunostaining of MMP-9 in brain sections from CAA patients (CAA) or control individuals (Control). Postmortem brain sections (5 μm/section, four sections per case) from the occipital lobar of CAA patients or control individuals were stained with an MMP-9 antibody recognizing the catalytic domain of MMP-9 (left panel, brown). CAA vessels were identified by thioflavin S staining (middle panel, green). The merged picture of MMP-9 staining and thioflavin S staining (right panel) shows the spatial association of MMP-9 (blue, pseudo color) and amyloid deposition (green). (B) The percentage of the thioflavin S positive vessels in total MMP-9 positive vessels. The proportion of thioflavin S positive vessels (Thio-S (+)) or thioflavin S negative vessels (Thio-S (−)) in all MMP-9 positive vessels was quantified with the CAST software. (C) Representative immunostaining of MMP-9 in CAA vessels at different stages or in control vessels. Postmortem brain sections from the occipital lobar of CAA patients or control individuals were stained with an MMP-9 antibody (upper panel, brown). CAA vessels were identified by thioflavin S staining (middle panel, green) and divided into five groups (S0, S1, S2, S3, S4) according to the grades of CAA severity as described in the Materials and Methods. The merged picture of MMP-9 staining and thioflavin S staining shows the spatial association of MMP-9 (blue, pseudo color) and amyloid deposition (green). (D) Quantitation of the MMP-9 immunoreactivity in CAA vessels at different stages or control vessels. The MMP-9 immunoreactivity of vessels was quantified by ImageJ. Vessels at stage 3 and stage 4 were combined to represent the advanced stage of CAA (S3/4). The immunoreactivity of vascular MMP-9 in brain sections from control cases were measured as the controls (Con.). *** p

    Journal: Neurobiology of aging

    Article Title: Matrix Metalloproteinase 9 mediated intracerebral hemorrhage induced by Cerebral amyloid angiopathy

    doi: 10.1016/j.neurobiolaging.2015.07.016

    Figure Lengend Snippet: MMP-9 accumulates in CAA-affected vessels (A) Representative immunostaining of MMP-9 in brain sections from CAA patients (CAA) or control individuals (Control). Postmortem brain sections (5 μm/section, four sections per case) from the occipital lobar of CAA patients or control individuals were stained with an MMP-9 antibody recognizing the catalytic domain of MMP-9 (left panel, brown). CAA vessels were identified by thioflavin S staining (middle panel, green). The merged picture of MMP-9 staining and thioflavin S staining (right panel) shows the spatial association of MMP-9 (blue, pseudo color) and amyloid deposition (green). (B) The percentage of the thioflavin S positive vessels in total MMP-9 positive vessels. The proportion of thioflavin S positive vessels (Thio-S (+)) or thioflavin S negative vessels (Thio-S (−)) in all MMP-9 positive vessels was quantified with the CAST software. (C) Representative immunostaining of MMP-9 in CAA vessels at different stages or in control vessels. Postmortem brain sections from the occipital lobar of CAA patients or control individuals were stained with an MMP-9 antibody (upper panel, brown). CAA vessels were identified by thioflavin S staining (middle panel, green) and divided into five groups (S0, S1, S2, S3, S4) according to the grades of CAA severity as described in the Materials and Methods. The merged picture of MMP-9 staining and thioflavin S staining shows the spatial association of MMP-9 (blue, pseudo color) and amyloid deposition (green). (D) Quantitation of the MMP-9 immunoreactivity in CAA vessels at different stages or control vessels. The MMP-9 immunoreactivity of vessels was quantified by ImageJ. Vessels at stage 3 and stage 4 were combined to represent the advanced stage of CAA (S3/4). The immunoreactivity of vascular MMP-9 in brain sections from control cases were measured as the controls (Con.). *** p

    Article Snippet: MMP-9 antibody (catalytic domain) and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Millipore; recombinant human MMP-9 (pre-activated) was purchased from Anaspec; monoclonal antibody against human fibrin (a′-fibrin) was purchased from American Diagnostic Inc.; The anti-fibrin antibody was labeled with DyLight 594 using a commercial kit (Thermo Scientific); Prussian blue staining kits were purchased from Polysciences, Inc.

    Techniques: Cellular Antioxidant Activity Assay, Immunostaining, Staining, Software, Quantitation Assay

    Real-time visualization and quantitation of lobar hemorrhage in vivo by multi-photon microscopy C57/BL6 mice, 3 months of age, were prepared with a craniotomy followed by removal of the dura mater. PBS or rMMP-9 (0.4 μg) was topically applied to the surface of mouse brains. Fluorescence-conjugated macromolecules were used as tracers. Animals were imaged hourly using a Fluoview 1000MPE Olympus multi-photon microscope to track the vascular leakage. The background images (BG) were acquired by imaging animals before administration of rMMP-9 and a tracer. (A) Examination of MMP-9-induced hemorrhage using fluorescent-anti-fibrin tracer. A mixture of a DyLight 594-conjugated anti-fibrin antibody (DyLight 594-α′-fibrin) and a fluorescein isothiocyanate-conjugated dextran (FITC-dextran, 70 kDa) was intravenously infused into tail veins of animals. Pre-activated rMMP-9 (0.4 μg) was topically applied to the surface of mouse brains followed by simultaneously imaging hourly in two channels to track the fluorescence signals of both DyLight 594-anti-fibrin (upper panel) and FITC-dextran (middle panel). The fluorescence signals of the DyLight 594-anti-fibrin or the FITC-dextran were merged to compare the detection sensitivity of the two tracers (lower panel). Arrows: fluorescent clots recognized by the fluorescent-anti-fibrin tracer. Scale bar = 100 μm. (B) The specificity of the fluorescent-anti-fibrin tracer in detecting of rMMP-9-induced lobar hemorrhage. Mice treated with 0.4 μg of rMMP-9 (middle and lower panels) or PBS (upper panel) were processed for hemorrhage assessment. DyLight 594-anti-fibrin (DyLight-a′-fibrin, upper and middle panels) or DyLight 594-conjuaged PHF1 antibody (DyLight-PHF1, lower panel) was used as a tracer. Arrows showing the fluorescent clots recognized by DyLight 594-a′-fibrin. Scale bar = 100 μm. (C) The volume of hemorrhage was determined by quantifying the fluorescence intensity of DyLight 594-anti-fibrin that accumulated at the clots. (D) Effects of different doses of rMMP-9 on hemorrhage induction. PBS (0 μg of rMMP-9) or rMMP-9 at dose of 0.05 μg, 0.2 μg, or 0.4 μg were topically applied to the mouse brains. Mice were processed for imaging hourly up to 7 hours of time period. DyLight 594-anti-fibrin was used as a tracer. The time that the clots were first detected (time to form clots) was measured. NS = not significant; ** p

    Journal: Neurobiology of aging

    Article Title: Matrix Metalloproteinase 9 mediated intracerebral hemorrhage induced by Cerebral amyloid angiopathy

    doi: 10.1016/j.neurobiolaging.2015.07.016

    Figure Lengend Snippet: Real-time visualization and quantitation of lobar hemorrhage in vivo by multi-photon microscopy C57/BL6 mice, 3 months of age, were prepared with a craniotomy followed by removal of the dura mater. PBS or rMMP-9 (0.4 μg) was topically applied to the surface of mouse brains. Fluorescence-conjugated macromolecules were used as tracers. Animals were imaged hourly using a Fluoview 1000MPE Olympus multi-photon microscope to track the vascular leakage. The background images (BG) were acquired by imaging animals before administration of rMMP-9 and a tracer. (A) Examination of MMP-9-induced hemorrhage using fluorescent-anti-fibrin tracer. A mixture of a DyLight 594-conjugated anti-fibrin antibody (DyLight 594-α′-fibrin) and a fluorescein isothiocyanate-conjugated dextran (FITC-dextran, 70 kDa) was intravenously infused into tail veins of animals. Pre-activated rMMP-9 (0.4 μg) was topically applied to the surface of mouse brains followed by simultaneously imaging hourly in two channels to track the fluorescence signals of both DyLight 594-anti-fibrin (upper panel) and FITC-dextran (middle panel). The fluorescence signals of the DyLight 594-anti-fibrin or the FITC-dextran were merged to compare the detection sensitivity of the two tracers (lower panel). Arrows: fluorescent clots recognized by the fluorescent-anti-fibrin tracer. Scale bar = 100 μm. (B) The specificity of the fluorescent-anti-fibrin tracer in detecting of rMMP-9-induced lobar hemorrhage. Mice treated with 0.4 μg of rMMP-9 (middle and lower panels) or PBS (upper panel) were processed for hemorrhage assessment. DyLight 594-anti-fibrin (DyLight-a′-fibrin, upper and middle panels) or DyLight 594-conjuaged PHF1 antibody (DyLight-PHF1, lower panel) was used as a tracer. Arrows showing the fluorescent clots recognized by DyLight 594-a′-fibrin. Scale bar = 100 μm. (C) The volume of hemorrhage was determined by quantifying the fluorescence intensity of DyLight 594-anti-fibrin that accumulated at the clots. (D) Effects of different doses of rMMP-9 on hemorrhage induction. PBS (0 μg of rMMP-9) or rMMP-9 at dose of 0.05 μg, 0.2 μg, or 0.4 μg were topically applied to the mouse brains. Mice were processed for imaging hourly up to 7 hours of time period. DyLight 594-anti-fibrin was used as a tracer. The time that the clots were first detected (time to form clots) was measured. NS = not significant; ** p

    Article Snippet: MMP-9 antibody (catalytic domain) and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Millipore; recombinant human MMP-9 (pre-activated) was purchased from Anaspec; monoclonal antibody against human fibrin (a′-fibrin) was purchased from American Diagnostic Inc.; The anti-fibrin antibody was labeled with DyLight 594 using a commercial kit (Thermo Scientific); Prussian blue staining kits were purchased from Polysciences, Inc.

    Techniques: Quantitation Assay, In Vivo, Microscopy, Mouse Assay, Fluorescence, Imaging

    MMP-9 induces an earlier onset and a greater magnitude of hemorrhage in Tg2576 mice compared to age-matched littermates (A) Hemorrhage induction of rMMP-9 in old Tg2576 mice. Tg2576 mice or their age-matched nontransgenic littermates, 15 months of age, were prepared with a craniotomy followed by removal of the dura mater. rMMP-9 (0.05 μg) was topically applied to brain surface of Tg2576 mice (Tg2576 − dura) or their age-matched nontransgenic littermates (WT - dura). Tg2576 mice that the dura mater was removed on the right hemispheres whereas the dura mater preserved on the left hemispheres were treated in parallel as a procedure control (Tg2576 + dura). Fluorescent-anti-fibrin was injected into mice via tail vein followed by imaging hourly up to 7 hours. Thirty minutes before imaging at 7 hour time point, 300 μl of methoxy-X04 was intraperitoneally injected into the animal to label CAA affected vessels (green). Arrows show the fluorescent clots recognized by fluorescent-anti-fibrin. Scale bar = 100 μm. (B) The time points that the clots were first detected within mouse brains (Time to bleed) were measured and compared. (C) The fluorescence intensity of fluorescent-anti-fibrin in sum intensity projection of stacks were measured and analyzed by ImageJ software. * p

    Journal: Neurobiology of aging

    Article Title: Matrix Metalloproteinase 9 mediated intracerebral hemorrhage induced by Cerebral amyloid angiopathy

    doi: 10.1016/j.neurobiolaging.2015.07.016

    Figure Lengend Snippet: MMP-9 induces an earlier onset and a greater magnitude of hemorrhage in Tg2576 mice compared to age-matched littermates (A) Hemorrhage induction of rMMP-9 in old Tg2576 mice. Tg2576 mice or their age-matched nontransgenic littermates, 15 months of age, were prepared with a craniotomy followed by removal of the dura mater. rMMP-9 (0.05 μg) was topically applied to brain surface of Tg2576 mice (Tg2576 − dura) or their age-matched nontransgenic littermates (WT - dura). Tg2576 mice that the dura mater was removed on the right hemispheres whereas the dura mater preserved on the left hemispheres were treated in parallel as a procedure control (Tg2576 + dura). Fluorescent-anti-fibrin was injected into mice via tail vein followed by imaging hourly up to 7 hours. Thirty minutes before imaging at 7 hour time point, 300 μl of methoxy-X04 was intraperitoneally injected into the animal to label CAA affected vessels (green). Arrows show the fluorescent clots recognized by fluorescent-anti-fibrin. Scale bar = 100 μm. (B) The time points that the clots were first detected within mouse brains (Time to bleed) were measured and compared. (C) The fluorescence intensity of fluorescent-anti-fibrin in sum intensity projection of stacks were measured and analyzed by ImageJ software. * p

    Article Snippet: MMP-9 antibody (catalytic domain) and anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Millipore; recombinant human MMP-9 (pre-activated) was purchased from Anaspec; monoclonal antibody against human fibrin (a′-fibrin) was purchased from American Diagnostic Inc.; The anti-fibrin antibody was labeled with DyLight 594 using a commercial kit (Thermo Scientific); Prussian blue staining kits were purchased from Polysciences, Inc.

    Techniques: Mouse Assay, Injection, Imaging, Cellular Antioxidant Activity Assay, Fluorescence, Software

    Mesenchymal leader cells in the 2D ex vivo injury culture model are enriched for molecules involved in invasion, CD44, MMP-2, and MMP-9. (A) Model of the different regions of the ex vivo mock cataract surgery explant that are separated by microdissection for this study from both top and side views. Cells found in the points of the star-shaped culture that contain the region of the lens that is occupied by epithelial cells in vivo are referred to as the original attachment zone (OAZ, yellow). In response to injury, cells move onto the denuded basement membrane (BM) into the central migration zone (CMZ, purple). (B–D) To examine whether there are migration-specific changes in expression of molecules associated with invasion after wounding, ex vivo injury explants were microdissected into OAZ and CMZ regions and extracted on days 1–3 for Western blot analysis for CD44, MMP-9, MMP-2, and GAPDH (loading control). Graphs depicting Western blot results from four independent studies show the relative expression of CD44, MMP-9, and MMP-2 to GAPDH. CD44, MMP-9, and MMP-2 were predominately expressed in the CMZ region. These molecules decreased in expression as wound healing progressed on day 2 and on day 3, when wound healing is typically completed. MMP-2 and MMP-9 were detected predominantly in their active forms (bottom arrows) compared with their inactive proenzymatic form (top arrows). For both MMP-2 and MMP-9, the active form (lower band) was quantified and is represented on the graphs as a ratio to GAPDH. Immunoblots were quantified using Kodak 1D software, and data were normalized to results at day 1 in the CMZ zone. Values for each graph and relative densities are plotted ±SEM. Representative Western blots are shown below each graph. (E–M) Ex vivo–wounded cultures were fixed and immunostained for vimentin (F, I, L; green) and colabeled for CD44 (E, G; red), MMP-9 (H, J; red), or MMP-2 (K, M; red). Vimentin-rich mesenchymal cells at the leading edge were enriched for MMP-2, MMP-9, and CD44. CD44 localized at cell–cell interfaces and to the tips of these cells at the leading edge (E–G, arrow). MMP-9 strongly localized to vimentin-rich cells, sometimes colocalized with vimentin (H–J, arrow) and also to regions in the area to be repaired just beyond the cells (H, J, arrowhead). Bar, 10 μm. (N) The role of MMP-2/9 function on wound healing in the 2D ex vivo injury cultures was evaluated using inhibitors to MMP-2, MMP-9, or MMP-2/9. Phase images were taken on days 0–3, from which the open wound area was calculated in at least six capsules and quantified using NIS Elements analysis software. Open wound area was plotted on the graphs ±SEM. Inhibition of MMP-2 and/or MMP-9 function resulted in only a slight delay in wound closure.

    Journal: Molecular Biology of the Cell

    Article Title: Cells activated for wound repair have the potential to direct collective invasion of an epithelium

    doi: 10.1091/mbc.E15-09-0615

    Figure Lengend Snippet: Mesenchymal leader cells in the 2D ex vivo injury culture model are enriched for molecules involved in invasion, CD44, MMP-2, and MMP-9. (A) Model of the different regions of the ex vivo mock cataract surgery explant that are separated by microdissection for this study from both top and side views. Cells found in the points of the star-shaped culture that contain the region of the lens that is occupied by epithelial cells in vivo are referred to as the original attachment zone (OAZ, yellow). In response to injury, cells move onto the denuded basement membrane (BM) into the central migration zone (CMZ, purple). (B–D) To examine whether there are migration-specific changes in expression of molecules associated with invasion after wounding, ex vivo injury explants were microdissected into OAZ and CMZ regions and extracted on days 1–3 for Western blot analysis for CD44, MMP-9, MMP-2, and GAPDH (loading control). Graphs depicting Western blot results from four independent studies show the relative expression of CD44, MMP-9, and MMP-2 to GAPDH. CD44, MMP-9, and MMP-2 were predominately expressed in the CMZ region. These molecules decreased in expression as wound healing progressed on day 2 and on day 3, when wound healing is typically completed. MMP-2 and MMP-9 were detected predominantly in their active forms (bottom arrows) compared with their inactive proenzymatic form (top arrows). For both MMP-2 and MMP-9, the active form (lower band) was quantified and is represented on the graphs as a ratio to GAPDH. Immunoblots were quantified using Kodak 1D software, and data were normalized to results at day 1 in the CMZ zone. Values for each graph and relative densities are plotted ±SEM. Representative Western blots are shown below each graph. (E–M) Ex vivo–wounded cultures were fixed and immunostained for vimentin (F, I, L; green) and colabeled for CD44 (E, G; red), MMP-9 (H, J; red), or MMP-2 (K, M; red). Vimentin-rich mesenchymal cells at the leading edge were enriched for MMP-2, MMP-9, and CD44. CD44 localized at cell–cell interfaces and to the tips of these cells at the leading edge (E–G, arrow). MMP-9 strongly localized to vimentin-rich cells, sometimes colocalized with vimentin (H–J, arrow) and also to regions in the area to be repaired just beyond the cells (H, J, arrowhead). Bar, 10 μm. (N) The role of MMP-2/9 function on wound healing in the 2D ex vivo injury cultures was evaluated using inhibitors to MMP-2, MMP-9, or MMP-2/9. Phase images were taken on days 0–3, from which the open wound area was calculated in at least six capsules and quantified using NIS Elements analysis software. Open wound area was plotted on the graphs ±SEM. Inhibition of MMP-2 and/or MMP-9 function resulted in only a slight delay in wound closure.

    Article Snippet: Antibodies used for Western blotting included CD44 (monoclonal antibody [mAb]; Developmental Studies Hybridoma Bank, Iowa City, IA), MMP-9 (polyclonal; Millipore), MMP-2 (monoclonal; Millipore), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Ex Vivo, Laser Capture Microdissection, In Vivo, Migration, Expressing, Western Blot, Software, Inhibition

    CD44/vimentin-rich mesenchymal cells that express markers of invadosomes are inherently invasive in Matrigel-Transwell chemoinvasion assays. (A–C) At 2 d in culture, cells that have invaded to the bottom of Matrigel-coated Transwell filters were coimmunostained for vimentin and CD44. Identification of these invaded cells as mesenchymal was determined by their positive labeling for both CD44 (A, C; red) and vimentin (B, C; green). Bar, 20 μM. (D–L) Cells that invaded in the Matrigel-Transwell chemoinvasion assays were labeled for characteristic invadosome molecules on day 2. Invaded cells were coimmunostained for the following combinations: cortactin (D, F; red) with Tks5 (E, F; green); MMP-2 (G, I; red) with MMP-9 (H, I; green); or cortactin (J, L; red) with MMP-9 (K, L; green). Invaded cells were rich in all invadopodia molecules examined, and regions of colabeling for cortactin and Tks5 (D–F, arrows), as well as cortactin and MMP-9 (J–L, arrows) were evident where these molecules may function in invadosomes for invasive function. Bar = 10 μm (D–I), 5 μm (J–L). (M, N) A Matrigel-Transwell chemoinvasion assay at 24 h in culture labeled for vimentin (green) and F-actin (blue) and also imaged by differential interference contrast (DIC). (M) A single vimentin-rich mesenchymal cell that had invaded through Matrigel to the bottom of the filter through a pore in the filter (seen as a hole in the DIC image; white arrow) is imaged en face, represented by the red arrow. Bar, 50 μm. (N) The same cell (red arrow) as shown in a 3D reconstruction side view of confocal Z -stacks collected from the explant basement membrane capsule of the inverted explant on top of the filter through to the cells that had invaded to the bottom of the filter. Here the transit of this cell through the filter pore (white arrow) is evident. Bar, 30 μM. Here A–C and M–N are Matrigel-Transwell chemoinvasion assays in which the explant on top of the filter was not removed before immunostaining. In D–L, the explant on top of the filter was removed before labeling.

    Journal: Molecular Biology of the Cell

    Article Title: Cells activated for wound repair have the potential to direct collective invasion of an epithelium

    doi: 10.1091/mbc.E15-09-0615

    Figure Lengend Snippet: CD44/vimentin-rich mesenchymal cells that express markers of invadosomes are inherently invasive in Matrigel-Transwell chemoinvasion assays. (A–C) At 2 d in culture, cells that have invaded to the bottom of Matrigel-coated Transwell filters were coimmunostained for vimentin and CD44. Identification of these invaded cells as mesenchymal was determined by their positive labeling for both CD44 (A, C; red) and vimentin (B, C; green). Bar, 20 μM. (D–L) Cells that invaded in the Matrigel-Transwell chemoinvasion assays were labeled for characteristic invadosome molecules on day 2. Invaded cells were coimmunostained for the following combinations: cortactin (D, F; red) with Tks5 (E, F; green); MMP-2 (G, I; red) with MMP-9 (H, I; green); or cortactin (J, L; red) with MMP-9 (K, L; green). Invaded cells were rich in all invadopodia molecules examined, and regions of colabeling for cortactin and Tks5 (D–F, arrows), as well as cortactin and MMP-9 (J–L, arrows) were evident where these molecules may function in invadosomes for invasive function. Bar = 10 μm (D–I), 5 μm (J–L). (M, N) A Matrigel-Transwell chemoinvasion assay at 24 h in culture labeled for vimentin (green) and F-actin (blue) and also imaged by differential interference contrast (DIC). (M) A single vimentin-rich mesenchymal cell that had invaded through Matrigel to the bottom of the filter through a pore in the filter (seen as a hole in the DIC image; white arrow) is imaged en face, represented by the red arrow. Bar, 50 μm. (N) The same cell (red arrow) as shown in a 3D reconstruction side view of confocal Z -stacks collected from the explant basement membrane capsule of the inverted explant on top of the filter through to the cells that had invaded to the bottom of the filter. Here the transit of this cell through the filter pore (white arrow) is evident. Bar, 30 μM. Here A–C and M–N are Matrigel-Transwell chemoinvasion assays in which the explant on top of the filter was not removed before immunostaining. In D–L, the explant on top of the filter was removed before labeling.

    Article Snippet: Antibodies used for Western blotting included CD44 (monoclonal antibody [mAb]; Developmental Studies Hybridoma Bank, Iowa City, IA), MMP-9 (polyclonal; Millipore), MMP-2 (monoclonal; Millipore), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Labeling, Immunostaining

    Functional properties of invading cells in response to injury. (A–G) The 3D Matrigel invasion assay of ex vivo–wounded explants at (A–D) day 6 in culture and (E–G) day 2 in culture. (A) A 3D reconstruction of a confocal Z -stack of cords of cells in the injury invasion assay labeled for vimentin (green) and F-actin (red), emphasizing the extension of vimentin-rich cells into the Matrigel matrix in leader-cell positions. Bar, 30 μm. (B, C) Cords of invading cells in the Matrigel invasion assay were immunolabeled for cortactin (red) and vimentin (green) and viewed by confocal microscopy. This projection image of a confocal Z -stack shows that the processes extended by the vimentin-rich mesenchymal cells into the Matrigel also expressed cortactin (red). Bar, 5 μm. (D) Labeling for MMP-9 (green) in cells at the leading edges of the cords extended into Matrigel and was distributed in discrete vesicular structures in the cells (F-actin; red) and also in a punctate pattern just beyond the cells in the Matrigel matrix, where it can act to degrade the matrix for invasion. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (E–G) Vimentin-rich cells at the invading front (green) also express αSMA (red), often with a punctate distribution (arrowhead). Labeling for F-actin (purple) was mostly absent from the areas with a strong distribution of αSMA puncta, suggesting that this actin population was nonfilamentous. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (H) The individual roles of MMP-2/9 and vimentin in invasion after wounding were evaluated using an inhibitor approach in the Matrigel-Transwell chemoinvasion assay. This study included the MMP-2 inhibitor MMP-2 i , the MMP-9 inhibitor MMP-9 i , the combined MMP-2/9 inhibitor MMP-2/9 i , and the vimentin inhibitor WFA. Invaded cells from at least 10 capsules were counted and quantified using MetaMorph or NIS Elements image analysis software. Relative numbers of invaded cells were plotted on graphs ±SEM. MMP-2 and MMP-9 function were required for invasion of cells in the Matrigel-Transwell assay. MMP-2 inhibited invasion by 99%, MMP-9 by 96%, and MMP2/9 by 84% in the Matrigel-Transwell assays. Inhibiting vimentin function with WFA suppressed invasion in the Matrigel-Transwell assay by 98%.

    Journal: Molecular Biology of the Cell

    Article Title: Cells activated for wound repair have the potential to direct collective invasion of an epithelium

    doi: 10.1091/mbc.E15-09-0615

    Figure Lengend Snippet: Functional properties of invading cells in response to injury. (A–G) The 3D Matrigel invasion assay of ex vivo–wounded explants at (A–D) day 6 in culture and (E–G) day 2 in culture. (A) A 3D reconstruction of a confocal Z -stack of cords of cells in the injury invasion assay labeled for vimentin (green) and F-actin (red), emphasizing the extension of vimentin-rich cells into the Matrigel matrix in leader-cell positions. Bar, 30 μm. (B, C) Cords of invading cells in the Matrigel invasion assay were immunolabeled for cortactin (red) and vimentin (green) and viewed by confocal microscopy. This projection image of a confocal Z -stack shows that the processes extended by the vimentin-rich mesenchymal cells into the Matrigel also expressed cortactin (red). Bar, 5 μm. (D) Labeling for MMP-9 (green) in cells at the leading edges of the cords extended into Matrigel and was distributed in discrete vesicular structures in the cells (F-actin; red) and also in a punctate pattern just beyond the cells in the Matrigel matrix, where it can act to degrade the matrix for invasion. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (E–G) Vimentin-rich cells at the invading front (green) also express αSMA (red), often with a punctate distribution (arrowhead). Labeling for F-actin (purple) was mostly absent from the areas with a strong distribution of αSMA puncta, suggesting that this actin population was nonfilamentous. A representative single optical plane captured by confocal microscopy. Bar, 10 μm. (H) The individual roles of MMP-2/9 and vimentin in invasion after wounding were evaluated using an inhibitor approach in the Matrigel-Transwell chemoinvasion assay. This study included the MMP-2 inhibitor MMP-2 i , the MMP-9 inhibitor MMP-9 i , the combined MMP-2/9 inhibitor MMP-2/9 i , and the vimentin inhibitor WFA. Invaded cells from at least 10 capsules were counted and quantified using MetaMorph or NIS Elements image analysis software. Relative numbers of invaded cells were plotted on graphs ±SEM. MMP-2 and MMP-9 function were required for invasion of cells in the Matrigel-Transwell assay. MMP-2 inhibited invasion by 99%, MMP-9 by 96%, and MMP2/9 by 84% in the Matrigel-Transwell assays. Inhibiting vimentin function with WFA suppressed invasion in the Matrigel-Transwell assay by 98%.

    Article Snippet: Antibodies used for Western blotting included CD44 (monoclonal antibody [mAb]; Developmental Studies Hybridoma Bank, Iowa City, IA), MMP-9 (polyclonal; Millipore), MMP-2 (monoclonal; Millipore), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Functional Assay, Invasion Assay, Ex Vivo, Labeling, Immunolabeling, Confocal Microscopy, Activated Clotting Time Assay, Software, Transwell Assay

    Tan-IIA inhibited migratory and invasive ability in human BCa cells. ( A ) Human BCa cells were treated with 0.2% DMSO as a vehicle control or 4 μg/mL Tan-IIA for 24 h and then seeded onto the transwell hanging insert for migration (24 h) and invasion (48 h) assays. Images were captured using an inverted microscope with 200× magnification; Scale bar: 50 μm. The migration and invasion of BCa cells were quantified by counting the stained cells that migrated into the underside of the hanging insert membrane; ( B ) human BCa cells were treated with different concentrations of Tan-IIA (1, 2 and 4 μg/mL) for 48 h. The protein of total cell lysates were then used to detect MMP-9/-2 protein expression using western blot, and the ( C ) supernatant was used to detect the enzymatic activity using zymography analysis. M: marker. Data are presented as means ± S.D. from three different experiments. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Tanshinone IIA Inhibits Epithelial-Mesenchymal Transition in Bladder Cancer Cells via Modulation of STAT3-CCL2 Signaling

    doi: 10.3390/ijms18081616

    Figure Lengend Snippet: Tan-IIA inhibited migratory and invasive ability in human BCa cells. ( A ) Human BCa cells were treated with 0.2% DMSO as a vehicle control or 4 μg/mL Tan-IIA for 24 h and then seeded onto the transwell hanging insert for migration (24 h) and invasion (48 h) assays. Images were captured using an inverted microscope with 200× magnification; Scale bar: 50 μm. The migration and invasion of BCa cells were quantified by counting the stained cells that migrated into the underside of the hanging insert membrane; ( B ) human BCa cells were treated with different concentrations of Tan-IIA (1, 2 and 4 μg/mL) for 48 h. The protein of total cell lysates were then used to detect MMP-9/-2 protein expression using western blot, and the ( C ) supernatant was used to detect the enzymatic activity using zymography analysis. M: marker. Data are presented as means ± S.D. from three different experiments. ** p

    Article Snippet: The antibodies against p-STAT3 (Tyr705), STAT3, E-cadherin, N-cadherin, Vimentin, Slug, Snail, MMP-2, MMP-9 and β-actin were all purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

    Techniques: BIA-KA, Migration, Inverted Microscopy, Staining, Expressing, Western Blot, Activity Assay, Zymography, Marker

    Effect of pioglitazone on MMP-9 activity and mRNA in 3T3-F442A adipocytes. A, 3T3-F442A adipocytes were treated with increasing concentrations pioglitazone (PIO; 1, 3, and 10 μ m ) in serum-free medium for 24 h, and MMP-9 activity was measured using

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Matrix Metalloproteinase-9 Is Increased in Obese Subjects and Decreases in Response to Pioglitazone

    doi: 10.1210/jc.2009-2623

    Figure Lengend Snippet: Effect of pioglitazone on MMP-9 activity and mRNA in 3T3-F442A adipocytes. A, 3T3-F442A adipocytes were treated with increasing concentrations pioglitazone (PIO; 1, 3, and 10 μ m ) in serum-free medium for 24 h, and MMP-9 activity was measured using

    Article Snippet: MMP-9 antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques: Activity Assay

    Effect of pioglitazone or metformin on MMP-9 expression. IGT subjects were treated with either pioglitazone (PIO; n = 20) or metformin (MET; n = 21) for 10 wk. MMP-9 mRNA was measured in adipose tissue by real-time PCR as described in Subjects and Methods

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Matrix Metalloproteinase-9 Is Increased in Obese Subjects and Decreases in Response to Pioglitazone

    doi: 10.1210/jc.2009-2623

    Figure Lengend Snippet: Effect of pioglitazone or metformin on MMP-9 expression. IGT subjects were treated with either pioglitazone (PIO; n = 20) or metformin (MET; n = 21) for 10 wk. MMP-9 mRNA was measured in adipose tissue by real-time PCR as described in Subjects and Methods

    Article Snippet: MMP-9 antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Dose-dependent effect of pioglitazone treatment on PKCα mRNA and effect of PKCα depletion on MMP-9 activity in 3T3-F442A adipocytes. A, Adipocytes were treated with increasing concentration of pioglitazone (PIO; 1, 3, and 10 μ

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Matrix Metalloproteinase-9 Is Increased in Obese Subjects and Decreases in Response to Pioglitazone

    doi: 10.1210/jc.2009-2623

    Figure Lengend Snippet: Dose-dependent effect of pioglitazone treatment on PKCα mRNA and effect of PKCα depletion on MMP-9 activity in 3T3-F442A adipocytes. A, Adipocytes were treated with increasing concentration of pioglitazone (PIO; 1, 3, and 10 μ

    Article Snippet: MMP-9 antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques: Activity Assay, Concentration Assay

    Effect of pioglitazone on MMP-9 protein and mRNA expression in THP1 macrophage cells. THP1 macrophages were differentiated as described in Subjects and Methods and treated with pioglitazone (PIO; 1.5 or 3 μ m ) for 24 h in serum-free medium. A,

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Matrix Metalloproteinase-9 Is Increased in Obese Subjects and Decreases in Response to Pioglitazone

    doi: 10.1210/jc.2009-2623

    Figure Lengend Snippet: Effect of pioglitazone on MMP-9 protein and mRNA expression in THP1 macrophage cells. THP1 macrophages were differentiated as described in Subjects and Methods and treated with pioglitazone (PIO; 1.5 or 3 μ m ) for 24 h in serum-free medium. A,

    Article Snippet: MMP-9 antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques: Expressing

    Adipose expression of MMP-9: correlation with BMI and S I . MMP-9 mRNA was quantitated in the adipose tissue of 86 subjects, as described in Subjects and Methods , by real-time RT-PCR and was expressed relative to endogenous 18S RNA. A, MMP-9 expression

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Matrix Metalloproteinase-9 Is Increased in Obese Subjects and Decreases in Response to Pioglitazone

    doi: 10.1210/jc.2009-2623

    Figure Lengend Snippet: Adipose expression of MMP-9: correlation with BMI and S I . MMP-9 mRNA was quantitated in the adipose tissue of 86 subjects, as described in Subjects and Methods , by real-time RT-PCR and was expressed relative to endogenous 18S RNA. A, MMP-9 expression

    Article Snippet: MMP-9 antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques: Expressing, Quantitative RT-PCR

    MMP-9 in adipocytes, macrophages, and cellular fractions of adipose tissue

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Matrix Metalloproteinase-9 Is Increased in Obese Subjects and Decreases in Response to Pioglitazone

    doi: 10.1210/jc.2009-2623

    Figure Lengend Snippet: MMP-9 in adipocytes, macrophages, and cellular fractions of adipose tissue

    Article Snippet: MMP-9 antibody was purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Techniques:

    Effects of the ERK inhibitor (U0126) and baicalein on cell invasion and MMP-2, MMP-9, u-PA, TIMP-1 and TIMP-2 expression in MHCC97H cells. (A) Cells were pretreated with U0126 (10 μM) for 30 min and then incubated in the presence or absence of baicalein (10 μM) for 24 h. Cellular invasiveness was measured using the Boyden chamber invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) MHCC97H cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9, u-PA, TIMP-1 and TIMP-2. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Journal: PLoS ONE

    Article Title: Baicalein Inhibits the Invasion and Metastatic Capabilities of Hepatocellular Carcinoma Cells via Down-Regulation of the ERK Pathway

    doi: 10.1371/journal.pone.0072927

    Figure Lengend Snippet: Effects of the ERK inhibitor (U0126) and baicalein on cell invasion and MMP-2, MMP-9, u-PA, TIMP-1 and TIMP-2 expression in MHCC97H cells. (A) Cells were pretreated with U0126 (10 μM) for 30 min and then incubated in the presence or absence of baicalein (10 μM) for 24 h. Cellular invasiveness was measured using the Boyden chamber invasion assay. (B) The percent invasion rate was expressed as a percentage of control. (C, D) MHCC97H cells were treated and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9, u-PA, TIMP-1 and TIMP-2. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Article Snippet: Anti-Phospho-MEK1 (Thr386) (p-MEK1) and anti-MEK1 were purchased from Millipore Co. Anti-Phospho (Thr202/Tyr204) ERK1/2 (p-ERK1/2), anti-ERK1/2, anti-MMP-2, anti-MMP-9, anti-TIMP-1 and anti-TIMP-2 antibodies were purchased from Cell Signaling.

    Techniques: Expressing, Incubation, Invasion Assay, Western Blot

    Baicalein suppresses the expression and activity of MMP-2, MMP-9 and u-PA and promotes the expression of TIMP-1 and TIMP-2 in MHCC97H cells. (A) The effects of baicalein on the expression levels of MMP-2, MMP-9 and u-PA mRNA were assessed by qRT-PCR. (B) MHCC97H cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9 and u-PA. (C) Quantification of the protein levels of MMP-2, MMP-9 and u-PA. (D) Effects of baicalein on the activities of MMP-2, MMP-9 and u-PA. (E) Quantification of the activities of MMP-2, MMP-9 and u-PA. (F) MHCC97H cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then subjected to western blotting to analyze the protein levels of TIMP-1 and TIMP-2. (G) Quantification of the protein levels of TIMP-1 and TIMP-2. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Journal: PLoS ONE

    Article Title: Baicalein Inhibits the Invasion and Metastatic Capabilities of Hepatocellular Carcinoma Cells via Down-Regulation of the ERK Pathway

    doi: 10.1371/journal.pone.0072927

    Figure Lengend Snippet: Baicalein suppresses the expression and activity of MMP-2, MMP-9 and u-PA and promotes the expression of TIMP-1 and TIMP-2 in MHCC97H cells. (A) The effects of baicalein on the expression levels of MMP-2, MMP-9 and u-PA mRNA were assessed by qRT-PCR. (B) MHCC97H cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2, MMP-9 and u-PA. (C) Quantification of the protein levels of MMP-2, MMP-9 and u-PA. (D) Effects of baicalein on the activities of MMP-2, MMP-9 and u-PA. (E) Quantification of the activities of MMP-2, MMP-9 and u-PA. (F) MHCC97H cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h and then subjected to western blotting to analyze the protein levels of TIMP-1 and TIMP-2. (G) Quantification of the protein levels of TIMP-1 and TIMP-2. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Article Snippet: Anti-Phospho-MEK1 (Thr386) (p-MEK1) and anti-MEK1 were purchased from Millipore Co. Anti-Phospho (Thr202/Tyr204) ERK1/2 (p-ERK1/2), anti-ERK1/2, anti-MMP-2, anti-MMP-9, anti-TIMP-1 and anti-TIMP-2 antibodies were purchased from Cell Signaling.

    Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot

    WEF reduces PMA-induced MMP-9 expression and MMP-9 activity. HT1080 cells pretreated with 25, 50, and 100 μg/ml WEF for 12 h in serum-free media were stimulated with 5 nM PMA for additional 24 h. (A): MMP-9 mRNA levels were measured by RT-PCR and relative expression was quantified after normalization to GAPDH. (B): CM were collected and analyzed for the MMP-9 level and MMP-9 activity by Western blotting and zymography, respectively. Gelatin and type I collagen were used as MMP-9 substrates. Data are expressed as the mean ± SD of two independent experiments. The full size blot was shown in the Supplementary Figure S3 and band of interest is indicated with an arrow. #, p

    Journal: Scientific Reports

    Article Title: Reduction of metastatic and angiogenic potency of malignant cancer by Eupatorium fortunei via suppression of MMP-9 activity and VEGF production

    doi: 10.1038/srep06994

    Figure Lengend Snippet: WEF reduces PMA-induced MMP-9 expression and MMP-9 activity. HT1080 cells pretreated with 25, 50, and 100 μg/ml WEF for 12 h in serum-free media were stimulated with 5 nM PMA for additional 24 h. (A): MMP-9 mRNA levels were measured by RT-PCR and relative expression was quantified after normalization to GAPDH. (B): CM were collected and analyzed for the MMP-9 level and MMP-9 activity by Western blotting and zymography, respectively. Gelatin and type I collagen were used as MMP-9 substrates. Data are expressed as the mean ± SD of two independent experiments. The full size blot was shown in the Supplementary Figure S3 and band of interest is indicated with an arrow. #, p

    Article Snippet: Antibodies against MMP-9, IκBα, pIκBα, NF-κB p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Akt, p-Akt, mTOR, p-mTOR, TBP, and tubulin were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Zymography

    HPV-16 E7 up-regulates MMP-9 activity in organotypic cultures. Primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. Cells were then differentiated in organotypic cultures for 9 to 11 days. A, Determination of gelatinase activity in organotypic cultures homogenates (epidermis separated from dermis). Equal amounts of proteins were loaded. Note the increase in MMP-9 gelatinase activity in HPV-16 E7wt and E6E7- expressing epidermis. B-B′, densitometry of pro-MMP-9 bands in epidermis and dermis, respectively. C, MMP-9 levels in epidermis homogenates were determined by Western blot. P

    Journal: PLoS ONE

    Article Title: HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

    doi: 10.1371/journal.pone.0033585

    Figure Lengend Snippet: HPV-16 E7 up-regulates MMP-9 activity in organotypic cultures. Primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. Cells were then differentiated in organotypic cultures for 9 to 11 days. A, Determination of gelatinase activity in organotypic cultures homogenates (epidermis separated from dermis). Equal amounts of proteins were loaded. Note the increase in MMP-9 gelatinase activity in HPV-16 E7wt and E6E7- expressing epidermis. B-B′, densitometry of pro-MMP-9 bands in epidermis and dermis, respectively. C, MMP-9 levels in epidermis homogenates were determined by Western blot. P

    Article Snippet: Measurement of MMP-2 and MMP-9 activity MMP-2 and MMP-9 activity was quantified in the culture supernatants from monolayer cultures of HFK transduced with retroviral vectors expressing HPV16 oncoproteins using specific Biotrak assay systems (MMP-2 Biotrak Activity Assay RPN 2631; MMP-9 Biotrak Activity Assay RPN2634, GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions.

    Techniques: Activity Assay, Transduction, Expressing, Western Blot

    Faecal MMP-9 levels in adenomas.

    Journal: British Journal of Cancer

    Article Title: A pilot study on faecal MMP-9: a new noninvasive diagnostic marker of colorectal cancer

    doi: 10.1038/bjc.2016.31

    Figure Lengend Snippet: Faecal MMP-9 levels in adenomas.

    Article Snippet: Active human MMP-9 full-length protein (Abcam LTD, Cambridge, UK; Cat. No.: ab157344; LOT No: GR218764-1) was added to the extraction medium.

    Techniques:

    Faecal MMP-9 levels in CRC patients. Values according to ( A ) localisation and ( B ) Dukes' stages of CRC.

    Journal: British Journal of Cancer

    Article Title: A pilot study on faecal MMP-9: a new noninvasive diagnostic marker of colorectal cancer

    doi: 10.1038/bjc.2016.31

    Figure Lengend Snippet: Faecal MMP-9 levels in CRC patients. Values according to ( A ) localisation and ( B ) Dukes' stages of CRC.

    Article Snippet: Active human MMP-9 full-length protein (Abcam LTD, Cambridge, UK; Cat. No.: ab157344; LOT No: GR218764-1) was added to the extraction medium.

    Techniques:

    MMP-9 concentration levels at different dilutions in the validation assay.

    Journal: British Journal of Cancer

    Article Title: A pilot study on faecal MMP-9: a new noninvasive diagnostic marker of colorectal cancer

    doi: 10.1038/bjc.2016.31

    Figure Lengend Snippet: MMP-9 concentration levels at different dilutions in the validation assay.

    Article Snippet: Active human MMP-9 full-length protein (Abcam LTD, Cambridge, UK; Cat. No.: ab157344; LOT No: GR218764-1) was added to the extraction medium.

    Techniques: Concentration Assay

    Faecal MMP-9 levels of the different groups (based on colonoscopy and histology). Abbreviation: CRC=colorectal carcinoma.

    Journal: British Journal of Cancer

    Article Title: A pilot study on faecal MMP-9: a new noninvasive diagnostic marker of colorectal cancer

    doi: 10.1038/bjc.2016.31

    Figure Lengend Snippet: Faecal MMP-9 levels of the different groups (based on colonoscopy and histology). Abbreviation: CRC=colorectal carcinoma.

    Article Snippet: Active human MMP-9 full-length protein (Abcam LTD, Cambridge, UK; Cat. No.: ab157344; LOT No: GR218764-1) was added to the extraction medium.

    Techniques:

    A model proposed for the cross-talk between N-cadherin cleavage and MMP-9 expression in NPC cell invasion

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: A model proposed for the cross-talk between N-cadherin cleavage and MMP-9 expression in NPC cell invasion

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Expressing

    PMA or CM1 upregulated MMP-9 via the ERK, p38 MAPK and NF-κB pathways. a PMA (m ϕ CM) induced MMP-9 expression via activation of MAPK and NF-κB pathways. NPC cells were pre-treated with specific inhibitors of ERK 1/2 (U0126, 10 μM), p38 (SB203580, 10 μM), JNK (SP600125, 10 μM) or NF-kB (QNZ, 10 μM) for 1 h, then treated with PMA (100 nM) or m ϕ CM for 18 h, and MMP-9 levels in the CM were determined by gelatin zymography. The relative level of MMP-9 expression was compared in cells treated with MAPK or NF-κB inhibitor plus PMA and PMA treatment alone; N = 3, * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: PMA or CM1 upregulated MMP-9 via the ERK, p38 MAPK and NF-κB pathways. a PMA (m ϕ CM) induced MMP-9 expression via activation of MAPK and NF-κB pathways. NPC cells were pre-treated with specific inhibitors of ERK 1/2 (U0126, 10 μM), p38 (SB203580, 10 μM), JNK (SP600125, 10 μM) or NF-kB (QNZ, 10 μM) for 1 h, then treated with PMA (100 nM) or m ϕ CM for 18 h, and MMP-9 levels in the CM were determined by gelatin zymography. The relative level of MMP-9 expression was compared in cells treated with MAPK or NF-κB inhibitor plus PMA and PMA treatment alone; N = 3, * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Expressing, Activation Assay, Zymography

    CM1-derived MMP-9 mediated cell invasion. C1 or CM1 was harvested as described in Fig. 7 . a NTF/N-cad was detected in CM1. Equal amounts of cell lysate or CM underwent western blot analysis with an anti-N-cadherin antibody (sc-7939) that recognizes the extracellular domain (FL/N-cad, ~130 kDa; NTF/N-cad, ~90 kDa). b Co-incubation with an anti-MMP-9 antibody decreased CM1-mediated cell invasion. CM1 was co-incubated with antibodies for IgG, MMP-9 or NTF/N-cad for 2 h, then introduced into the outer well of the Boyden chamber for 24 h. Cells on the lower surface of the membrane were fixed and stained with crystal violet. Data are mean±SEM. N = 3, * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: CM1-derived MMP-9 mediated cell invasion. C1 or CM1 was harvested as described in Fig. 7 . a NTF/N-cad was detected in CM1. Equal amounts of cell lysate or CM underwent western blot analysis with an anti-N-cadherin antibody (sc-7939) that recognizes the extracellular domain (FL/N-cad, ~130 kDa; NTF/N-cad, ~90 kDa). b Co-incubation with an anti-MMP-9 antibody decreased CM1-mediated cell invasion. CM1 was co-incubated with antibodies for IgG, MMP-9 or NTF/N-cad for 2 h, then introduced into the outer well of the Boyden chamber for 24 h. Cells on the lower surface of the membrane were fixed and stained with crystal violet. Data are mean±SEM. N = 3, * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Derivative Assay, Western Blot, Incubation, Staining

    Cross-talk regulation between N-cadherin cleavage and MMP-9 expression. a SiRNA silencing of N-cadherin disrupted the cell–cell adhesion. NPC cells were transfected with N-cadherin siRNA for 24 h, then examined by immunofluoresence staining with anti-N-cadherin antibodies and analyzed by confocal laser scanning microscopy. b Silencing of N-cadherin decreased PMA-mediated cell invasion. NPC cells were transfected with N-cadherin siRNA for 24 h, then cell invasion was investigated by Boyden chamber assay. Representative images are shown and data are presented as mean±SEM. c NPC cells were transfected with siRNA specific for MMP-9 or N-cadherin for 24 h, then exposed to PMA for 24 h. The transfected cells were cultured with and without PMA for 24 h. The expression of MMP-9 and N-cadherin in cell lysates was assessed by western blot analysis with the indicated antibodies. MMP-9 level in CM was detected by gelatin zymography. The relative expression of FL/N-cad or MMP-9 in MMP-9 siRNA- or N-cadsiRNA-transfected cells was compared with Ng (non-specific control) siRNA (Ngi) -transfected cells after PMA treatment; N = 3, * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: Cross-talk regulation between N-cadherin cleavage and MMP-9 expression. a SiRNA silencing of N-cadherin disrupted the cell–cell adhesion. NPC cells were transfected with N-cadherin siRNA for 24 h, then examined by immunofluoresence staining with anti-N-cadherin antibodies and analyzed by confocal laser scanning microscopy. b Silencing of N-cadherin decreased PMA-mediated cell invasion. NPC cells were transfected with N-cadherin siRNA for 24 h, then cell invasion was investigated by Boyden chamber assay. Representative images are shown and data are presented as mean±SEM. c NPC cells were transfected with siRNA specific for MMP-9 or N-cadherin for 24 h, then exposed to PMA for 24 h. The transfected cells were cultured with and without PMA for 24 h. The expression of MMP-9 and N-cadherin in cell lysates was assessed by western blot analysis with the indicated antibodies. MMP-9 level in CM was detected by gelatin zymography. The relative expression of FL/N-cad or MMP-9 in MMP-9 siRNA- or N-cadsiRNA-transfected cells was compared with Ng (non-specific control) siRNA (Ngi) -transfected cells after PMA treatment; N = 3, * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Expressing, Transfection, Staining, Confocal Laser Scanning Microscopy, Boyden Chamber Assay, Cell Culture, Western Blot, Zymography

    CM1 significantly enhanced cell invasion accompanied by increments of MMP-9 upregulation and N-cadherin cleavage. Briefly, NPC cells were cultured with and without PMA for 8 h, then PMA-containing medium was completely removed and replaced with completed medium for 24 h. The CM was collected and referred to as CM1 or C1. a CM1 induced NPC cell invasion. The invasive capability of CM1-treated NPC cells was investigated. CM1 was introduced into the outer well of NPC-cell–seeded Boyden chambers for 24 h. The invasive cells at the lower surface of the membrane were fixed, stained, photographed and counted. Data are mean±SEM. * p

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: CM1 significantly enhanced cell invasion accompanied by increments of MMP-9 upregulation and N-cadherin cleavage. Briefly, NPC cells were cultured with and without PMA for 8 h, then PMA-containing medium was completely removed and replaced with completed medium for 24 h. The CM was collected and referred to as CM1 or C1. a CM1 induced NPC cell invasion. The invasive capability of CM1-treated NPC cells was investigated. CM1 was introduced into the outer well of NPC-cell–seeded Boyden chambers for 24 h. The invasive cells at the lower surface of the membrane were fixed, stained, photographed and counted. Data are mean±SEM. * p

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Cell Culture, Staining

    Blockade of N-cadherin cleavage decreased m ϕ CM-induced cell invasion and MMP-9 levels. a m ϕ CM enhanced NPC cell invasion. NPC cells were seeded into the inner well of the Boyden chamber, pre-coated with matrix-gel and monoCM or m ϕ CM, then introduced into the outer well of the Boyden chamber for 24 h. Cells that invaded the lower surface of the filter were fixed, stained, photographed and counted. Representative plots of Matrigel invasion assay are shown. Data are mean±SEM. N = 3, * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: Blockade of N-cadherin cleavage decreased m ϕ CM-induced cell invasion and MMP-9 levels. a m ϕ CM enhanced NPC cell invasion. NPC cells were seeded into the inner well of the Boyden chamber, pre-coated with matrix-gel and monoCM or m ϕ CM, then introduced into the outer well of the Boyden chamber for 24 h. Cells that invaded the lower surface of the filter were fixed, stained, photographed and counted. Representative plots of Matrigel invasion assay are shown. Data are mean±SEM. N = 3, * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Staining, Invasion Assay

    PMA induced the cleavage of N-cadherin. a N-cad and MMP9 expression with/without PMA treatment. NPC cells were exposed to PMA at the indicated times, and cell lysates underwent immunoblotting. The relative band intensities of N-cadherin and MMP-9 proteins were normalized to GAPDH by densitometry. Data are mean±SEM. * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: PMA induced the cleavage of N-cadherin. a N-cad and MMP9 expression with/without PMA treatment. NPC cells were exposed to PMA at the indicated times, and cell lysates underwent immunoblotting. The relative band intensities of N-cadherin and MMP-9 proteins were normalized to GAPDH by densitometry. Data are mean±SEM. * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Expressing

    MMP-9 inhibitors abolished PMA-inducedN-cadherin cleavage. NPC cells were pre-treated with ( a ) GM6001 (GM, 10–20 μM) or ( b ) a specific MMP-9 inhibitor (20 μM) for 2 h, then co-incubated with PMA (100 nM) for 24 h. Cell lysates underwent immunoblotting with the indicated antibodies. The relative band intensities of FL/N-cad were compared with the control group (PMA-untreated cells), and relative expression (RLE) of N-cadherin is shown as the ratio of GM-plus-PMA–treated group to PMA treatment alone. c The effect of pro-MMP-9 or activated MMP-9 on the cleavage of N-cadherin.Pro-MMP-9 (R D, 911-MPN-010) was activated by incubation in TCNB buffer for 24 h at 37 °C. NPC-TW039 cells were exposed to activated MMP-9 or pro-MMP-9 (0.1 nM) for 7 h. Cell lysates underwent western blot analysis with anti-N-cadherin antibody. The relative expression of FL/N-cad was compared to the control group (activated MMP-9-untreated cells); N = 3, * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: MMP-9 inhibitors abolished PMA-inducedN-cadherin cleavage. NPC cells were pre-treated with ( a ) GM6001 (GM, 10–20 μM) or ( b ) a specific MMP-9 inhibitor (20 μM) for 2 h, then co-incubated with PMA (100 nM) for 24 h. Cell lysates underwent immunoblotting with the indicated antibodies. The relative band intensities of FL/N-cad were compared with the control group (PMA-untreated cells), and relative expression (RLE) of N-cadherin is shown as the ratio of GM-plus-PMA–treated group to PMA treatment alone. c The effect of pro-MMP-9 or activated MMP-9 on the cleavage of N-cadherin.Pro-MMP-9 (R D, 911-MPN-010) was activated by incubation in TCNB buffer for 24 h at 37 °C. NPC-TW039 cells were exposed to activated MMP-9 or pro-MMP-9 (0.1 nM) for 7 h. Cell lysates underwent western blot analysis with anti-N-cadherin antibody. The relative expression of FL/N-cad was compared to the control group (activated MMP-9-untreated cells); N = 3, * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Incubation, Expressing, Western Blot

    Blockade of N-cadherin cleavage decreased PMA-mediated cell invasion and MMP-9 level. a γI treatment decreased PMA-induced cell invasion. NPC cells were treated with γI in the inner well of the Boyden chamber, and PMA (100 nM) plus γI was introduced into the outer well for 24 h. Cells that invaded the lower surface of the filter were fixed, stained and photographed. Representative images and statistical plots of the invasion assay are shown. The relative cell invasion in the γI-plus-PMA–treated group was compared to PMA treatment alone. b γI treatment decreased the PMA-mediatedMMP-9 expression. NPC cells were pre-treated with γI (5 μM), then exposed to PMA for 24 h. Cell lysates underwent immunoblotting with the indicated antibodies. The relative MMP-9 expression was compared with the control group (PMA-untreated cells); N = 3, * P

    Journal: BMC Cancer

    Article Title: Interplay of N-Cadherin and matrix metalloproteinase 9 enhances human nasopharyngeal carcinoma cell invasion

    doi: 10.1186/s12885-016-2846-4

    Figure Lengend Snippet: Blockade of N-cadherin cleavage decreased PMA-mediated cell invasion and MMP-9 level. a γI treatment decreased PMA-induced cell invasion. NPC cells were treated with γI in the inner well of the Boyden chamber, and PMA (100 nM) plus γI was introduced into the outer well for 24 h. Cells that invaded the lower surface of the filter were fixed, stained and photographed. Representative images and statistical plots of the invasion assay are shown. The relative cell invasion in the γI-plus-PMA–treated group was compared to PMA treatment alone. b γI treatment decreased the PMA-mediatedMMP-9 expression. NPC cells were pre-treated with γI (5 μM), then exposed to PMA for 24 h. Cell lysates underwent immunoblotting with the indicated antibodies. The relative MMP-9 expression was compared with the control group (PMA-untreated cells); N = 3, * P

    Article Snippet: The anti-MMP-9 antibody used for neutralizing MMP-9 activities in the conditioned medium and for western blotting was purchased from Epitomics.

    Techniques: Staining, Invasion Assay, Expressing

    TXA reduces activation of MMP-2 and MMP-9 in the injured spinal cord. Contusion SCI was induced by the Infinite Horizons impactor in C57BL/6 mice treated without or with TXA. Mice were treated with a bolus intravenous injection of TXA just after SCI followed by per os administration of TXA (20 mg/mL of drinking water). The concentrations of active MMP-2 ( a ) and MMP-9 ( b ) in the spinal cord were assessed at day 0 (laminectomised mice without SCI; Sham), day 1 (1 dpi), or day 7 (7 dpi). Values and error bars represent mean ± SD ( n = 6 in each group). * P

    Journal: Scientific Reports

    Article Title: Short-term inhibition of fibrinolytic system restores locomotor function after spinal cord injury in mice

    doi: 10.1038/s41598-019-52621-8

    Figure Lengend Snippet: TXA reduces activation of MMP-2 and MMP-9 in the injured spinal cord. Contusion SCI was induced by the Infinite Horizons impactor in C57BL/6 mice treated without or with TXA. Mice were treated with a bolus intravenous injection of TXA just after SCI followed by per os administration of TXA (20 mg/mL of drinking water). The concentrations of active MMP-2 ( a ) and MMP-9 ( b ) in the spinal cord were assessed at day 0 (laminectomised mice without SCI; Sham), day 1 (1 dpi), or day 7 (7 dpi). Values and error bars represent mean ± SD ( n = 6 in each group). * P

    Article Snippet: Dissected spinal cords were homogenised in Tris-HCl buffer containing 0.1% Triton-X-100, and assessed using mouse MMP-2 and MMP-9 activity assays (QuickZyme Biosciences, Leiden, the Netherlands).

    Techniques: Activation Assay, Mouse Assay, Injection

    Effect of inhibitors on the expression of MMP-9 mRNA. To investigate which cellular signaling pathways were associated with the upregulation of MMP-9 following treatment with arecoline, inhibitors of signaling pathway were used, including the NF-κB/IκB

    Journal: Oncology Letters

    Article Title: Upregulated expression of MMP-9 in gingival epithelial cells induced by prolonged stimulation with arecoline

    doi: 10.3892/ol.2017.6194

    Figure Lengend Snippet: Effect of inhibitors on the expression of MMP-9 mRNA. To investigate which cellular signaling pathways were associated with the upregulation of MMP-9 following treatment with arecoline, inhibitors of signaling pathway were used, including the NF-κB/IκB

    Article Snippet: MMP-2 and MMP-9 activity were measured using an MMP-2 and MMP-9 activity assay kits (QuickZyme Biosciences, Leiden, Netherlands) according to the manufacturer's protocol.

    Techniques: Expressing

    MMP-2 and MMP-9 activity assays in the supernatant of cultured HGEPs. MMP-2 and MMP-9 activity were assessed with MMP-2 and MMP-9 activity assays. The supernatant was isolated from HGEPs treated with arecoline for 18 or 30 days. The supernatant without

    Journal: Oncology Letters

    Article Title: Upregulated expression of MMP-9 in gingival epithelial cells induced by prolonged stimulation with arecoline

    doi: 10.3892/ol.2017.6194

    Figure Lengend Snippet: MMP-2 and MMP-9 activity assays in the supernatant of cultured HGEPs. MMP-2 and MMP-9 activity were assessed with MMP-2 and MMP-9 activity assays. The supernatant was isolated from HGEPs treated with arecoline for 18 or 30 days. The supernatant without

    Article Snippet: MMP-2 and MMP-9 activity were measured using an MMP-2 and MMP-9 activity assay kits (QuickZyme Biosciences, Leiden, Netherlands) according to the manufacturer's protocol.

    Techniques: Activity Assay, Cell Culture, Isolation

    Expression level of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA in HGEPs. (A) No significant differences in the expression of MMP-2 mRNA were observed between the experimental and control groups. (B) The expression of MMP-9 mRNA in the experimental group was

    Journal: Oncology Letters

    Article Title: Upregulated expression of MMP-9 in gingival epithelial cells induced by prolonged stimulation with arecoline

    doi: 10.3892/ol.2017.6194

    Figure Lengend Snippet: Expression level of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA in HGEPs. (A) No significant differences in the expression of MMP-2 mRNA were observed between the experimental and control groups. (B) The expression of MMP-9 mRNA in the experimental group was

    Article Snippet: MMP-2 and MMP-9 activity were measured using an MMP-2 and MMP-9 activity assay kits (QuickZyme Biosciences, Leiden, Netherlands) according to the manufacturer's protocol.

    Techniques: Expressing