heparinase i Search Results


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  • 94
    New England Biolabs bacteroides heparinase i
    Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides <t>Heparinase</t> I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.
    Bacteroides Heparinase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore heparinase i
    LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither <t>heparinase</t> ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.
    Heparinase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore heparinase iii
    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without <t>heparinase</t> <t>III</t> for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p
    Heparinase Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 760 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore heparinase ii
    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without <t>heparinase</t> <t>III</t> for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p
    Heparinase Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore flavobacterium heparinum
    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase <t>I/III</t> blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P
    Flavobacterium Heparinum, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Seikagaku heparinase
    N-sulfate-enriched microenvironment forms a belt around the demyelinated lesion. HS (10E4) labeling on the contra- ( A ) and ipsi- ( B–C ) lateral side to the lesion illustrates the generation of a N-sulfated microenvironment surrounding the lesion (delimited by white dashed lines) at five dpi (n = 3). No immunoreactivity was found after <t>Heparinase</t> I treatment ( D ) thus validating the 10E4 antibody specificity. Scale bars: 20 µm in A, B, D; 10 µm in C. CC, corpus callosum; V, ventricle.
    Heparinase, supplied by Seikagaku, used in various techniques. Bioz Stars score: 91/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    IBEX Technologies heparinase iii
    Effects of PD98059 and <t>heparinase</t> on SMC marker gene expression under 3-D interstitial flow. PD98059 and heparinase reverse interstitial flow-induced reductions in SM-MHC, smoothelin, and calponin expression, but further enhance α-SMA and SM22 expression in 3-D. SMCs (A) and MFBs (B) in collagen gels were pretreated with PD98059 (PD) or heparinase <t>III</t> (Hepr) for 3 h, and then exposed to interstitial flow (1 cmH 2 O) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P
    Heparinase Iii, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Mimetics heparanase
    NK cell–intrinsic <t>heparanase</t> is indispensable for efficient surveillance of MCA-induced fibrosarcoma and RAE-1–expressing lymphoma. ( A – C ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were inoculated s.c. in the hind flank with 100 μg MCA in 0.1 ml corn oil. Mice were then monitored over a 200-day period for fibrosarcoma development. Tumors were measured every week with a caliper ( n = 24–28 per group; data were pooled from 2 independent experiments). ( A ) Data were recorded as the percentage of tumor-free mice (tumors were defined as measuring > 3 mm in diameter and consistently growing). Tumor growth curves of individual ( B ) Hpse fl/fl NKp46-WT and ( C ) Hpse fl/fl NKp46-iCre mice. ( D ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected s.c. with 5 × 10 6 RMA-S-RAE-1β cells. Tumor growth was measured every 2 to 3 days with a caliper (mean ± SEM; n = 7 per group; 1 representative experiment of 2 experiments). Statistically significant differences were determined by log-rank Mantel-Cox test ( A ) and Mann-Whitney U test ( D ). * P
    Heparanase, supplied by Mimetics, used in various techniques. Bioz Stars score: 91/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Seikagaku heparinase iii
    GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K d s were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125 I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with <t>heparinase</t> <t>III</t> (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125 I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.
    Heparinase Iii, supplied by Seikagaku, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    IBEX Technologies heparinase i
    Both HS and protein components of SCM are required for boundary formation Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); <t>heparinase</t> treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75 NTR (green). The number of cells mingling with astrocytes was counted across a 300 μm line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate ± SEM. Scale bar 50 μm. ** p
    Heparinase I, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Mimetics heparanase inhibitors
    ( A ) Overview of the <t>heparanase–GlcA</t> complex structure. The 8 and 50 kDa subunits are depicted with yellow and purple carbons, respectively, while the arrows indicate the location of the GS3 peptide. The TIM-barrel and β-sandwich domains of heparanase are contoured with red and green lines, respectively. GlcA is represented as spheres with white carbons. ( B ) Close-up view of the catalytic site of heparanase (white cartoons and carbons). The two catalytic residues are colored in yellow and GlcA in green.
    Heparanase Inhibitors, supplied by Mimetics, used in various techniques. Bioz Stars score: 88/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson heparinised vacutainer tubes
    ( A ) Overview of the <t>heparanase–GlcA</t> complex structure. The 8 and 50 kDa subunits are depicted with yellow and purple carbons, respectively, while the arrows indicate the location of the GS3 peptide. The TIM-barrel and β-sandwich domains of heparanase are contoured with red and green lines, respectively. GlcA is represented as spheres with white carbons. ( B ) Close-up view of the catalytic site of heparanase (white cartoons and carbons). The two catalytic residues are colored in yellow and GlcA in green.
    Heparinised Vacutainer Tubes, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    IBEX Technologies heparin lyases
    ( A ) Overview of the <t>heparanase–GlcA</t> complex structure. The 8 and 50 kDa subunits are depicted with yellow and purple carbons, respectively, while the arrows indicate the location of the GS3 peptide. The TIM-barrel and β-sandwich domains of heparanase are contoured with red and green lines, respectively. GlcA is represented as spheres with white carbons. ( B ) Close-up view of the catalytic site of heparanase (white cartoons and carbons). The two catalytic residues are colored in yellow and GlcA in green.
    Heparin Lyases, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems heparinase iii
    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and <t>Heparinase</t> <t>III.</t> Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Heparinase Iii, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Seikagaku heparin lyases
    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and <t>Heparinase</t> <t>III.</t> Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.
    Heparin Lyases, supplied by Seikagaku, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology heparanase
    Pancreas inflammation, cytokine induction and signaling pathways evoked by cerulein are attenuated by <t>heparanase</t> inhibitor. WT and Hpa-Tg mice were injected with saline (left panels) or cerulein in the absence (middle panels) or presence (right panels) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for neutrophils infiltration ( A ), p65 ( D ) and phospho-STAT3 ( F ). Immunofluorescent staining of corresponding sections for TNFα (green) is shown in ( C ) along with nuclear counterstaining (blue). Shown are representative photomicrographs at x10 ( A ) and x40 ( C,D,F ) original magnification. Total RNA was extracted from corresponding pancreas tissues and subjected to real-time PCR analyses applying primers specific for TNFα ( B ) and IL-6 ( E ). Relative gene expression (fold-increase) is shown graphically in relation to the levels in control pancreas set arbitrarily to a value of 1. *p
    Heparanase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti heparanase
    hTERT regulates the transcriptional activity of <t>heparanase</t> through the c-Myc binding site A. The 5′-flanking promoter region of the heparanase gene was cloned into the NheI/HindIII site of the luciferase construct pGL3-Basic vector. B. The relative luciferase activities of the heparanase promoter were tested using the dual luciferase assay in SGC7901 and MKN45 cells that were transiently transfected with pIRES2-hTERT (50, 200, 500 ng) and sh-hTERT-1 and sh-hTERT-2 vectors, respectively. C. The putative binding site of c-Myc on the heparanase promoter region was predicted using bioinformatics analysis. D. Chromatin immunoprecipitation (ChIP) assays were conducted in SGC7901 and MKN45 cells using an antibody against c-Myc or a control rabbit normal immunoglobulin G. An equivalent of amount of DNA in all of the samples served as an Input control. E. The luciferase activities of the heparanase promoter with wild type (or the mutant binding site of c-Myc) were determined using luciferase reporter assays in SGC7901 and MKN45 cells that were transiently transfected with pIRES2, pIRES2-hTERT plasmids. (* P
    Anti Heparanase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems heparanase
    Structures docked into human <t>heparanase:</t> (A) diantennary monomer 6 , (B) monoantennary monomer 5 , (C) hydrogen bonding network of disaccharide of dianntennary monomer 6 in heparanase subsites −1/−2, (D) hydrogen boding monosaccharide of dianntennary monomer 6 to HBD-2, and (E) hydrogen bonding network of disaccharide of monoanntennary monomer 5 in heparanase subsites −1/−2. Hydrogen bonding shown in magenta. HBD-2 is on the left and HBD-1 is on the right.
    Heparanase, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ImClone anti heparanase monoclonal antibody
    Comparison of splice 5 (S5) and wild-type (WT) human <t>heparanase</t> amino acid sequences (GCG alignment program). The two catalytic Glu residues, the proton donor and nucleophile, are marked in bold. Note that the proton donor is missing in splice 5. Potential
    Anti Heparanase Monoclonal Antibody, supplied by ImClone, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Journal: Scientific Reports

    Article Title: Heparin affinity purification of extracellular vesicles

    doi: 10.1038/srep10266

    Figure Lengend Snippet: Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Article Snippet: Beads were then washed three times with PBS 1x and either treated with Bacteroides Heparinase I (New England Biolabs, Ipswich, MA; 60U/ml heparin beads) according to the manufacturer’s recommendations or mock treated (incubation buffer devoid of heparinase) for 1 hr at 30 °C.

    Techniques: Isolation, Purification, Incubation, Biomarker Assay, Derivative Assay

    LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.

    Journal: Glycobiology

    Article Title: LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

    doi: 10.1093/glycob/cww075

    Figure Lengend Snippet: LARGE2 can modify GPC4 with the laminin-binding glycan. ( A ) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. ( B ) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. ( C ) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase ( D ) nor aqHF ( E ) removed the functional modification from GPC4Fc.

    Article Snippet: Heparinase treatment and HF dephosphorylation For heparinase treatment, samples were treated with a mixture of heparinases I and III (Sigma, 0.6 and 0.3 Sigma units, respectively) in 0.

    Techniques: Binding Assay, Construct, Sequencing, Purification, Expressing, Functional Assay, Modification

    Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced intracellular reactive oxygen species (ROS) production. ( a ) Quantitative results of ROS levels in each group according to (b). ( b ) Representative profiles of the intracellular ROS levels detected by flow cytometry using the 2’,7’-dichlorofluoroescin diacetate (DCFH-DA) assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Flow Cytometry, DCFH-DA Assay, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in SH-SY5Y cell morphology and cell viability. ( a ) SH-SY5Y cell morphology. Bar, 10 μM. Images were analyzed with SPOT 4.7 Advanced software. The arrows indicate the shorter neurite outgrowth of SH-SY5Y cells after the Aβ(25-35) challenge. ( b ) SH-SY5Y cell viability was analyzed by an MTT assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in SH-SY5Y cell morphology and cell viability. ( a ) SH-SY5Y cell morphology. Bar, 10 μM. Images were analyzed with SPOT 4.7 Advanced software. The arrows indicate the shorter neurite outgrowth of SH-SY5Y cells after the Aβ(25-35) challenge. ( b ) SH-SY5Y cell viability was analyzed by an MTT assay. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Software, MTT Assay, Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in the phosphorylated (p)-tau/tau ratio ( a ), phosphorylated (p)-phosphatidylinositol 3K (PI3K)/PI3K ratio ( b ), the phosphorylated (p)-Akt/Akt ratio ( c ), and the phosphorylated (p)-glycogen synthase kinase (GSK)-3β (Ser 9 )/GSK-3β ratio ( d ) of protein expressions. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in the phosphorylated (p)-tau/tau ratio ( a ), phosphorylated (p)-phosphatidylinositol 3K (PI3K)/PI3K ratio ( b ), the phosphorylated (p)-Akt/Akt ratio ( c ), and the phosphorylated (p)-glycogen synthase kinase (GSK)-3β (Ser 9 )/GSK-3β ratio ( d ) of protein expressions. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments, and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Incubation

    Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in cell apoptosis. ( a ) Western blot analysis of caspase-3 protein expression in SH-SY5Y cells. ( b ) Percentages of apoptotic cells in each group quantified from ( c ). ( c ) Representative profiles of cell apoptosis detected by flow cytometry with Annexin V/propidium iodide double-staining. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Journal: International Journal of Molecular Sciences

    Article Title: 1,25(OH)2D3 Alleviates Aβ(25-35)-Induced Tau Hyperphosphorylation, Excessive Reactive Oxygen Species, and Apoptosis Through Interplay with Glial Cell Line-Derived Neurotrophic Factor Signaling in SH-SY5Y Cells

    doi: 10.3390/ijms21124215

    Figure Lengend Snippet: Effects of 1,25(OH) 2 D 3 on Aβ-induced changes in cell apoptosis. ( a ) Western blot analysis of caspase-3 protein expression in SH-SY5Y cells. ( b ) Percentages of apoptotic cells in each group quantified from ( c ). ( c ) Representative profiles of cell apoptosis detected by flow cytometry with Annexin V/propidium iodide double-staining. SH-SY5Y cells were incubated with 1 μM Aβ(25-35) prior to the addition of 0.1 and 10 nM 1,25(OH) 2 D 3 with or without heparinase III for 24 h. Data are presented as the mean ± SD of three experiments and each experiment included triplicate repeats. * ,+,# Significantly differs between the two groups (statistical analysis was performed using Student’s t test). Bars of Aβ, Aβ + 0.1 nM 1,25(OH) 2 D 3 , and Aβ + 10 nM 1,25(OH) 2 D 3 with different letters significantly differ ( p

    Article Snippet: Then, cells were incubated with 1 μM of Aβ(25-35) for 24 h, followed by washing and incubation with two different concentrations of 1,25(OH)2 D3 (0.1 or 10nM) for 24 h. Heparinase III (H8891, Sigma, St. Louis, MO, USA), an inhibitor of the GDNF-signaling, was used with 1,25(OH)2 D3 treatment in some of the experiments to elucidate the role of GDNF on 1,25(OH)2 D3-stimulated effects.

    Techniques: Western Blot, Expressing, Flow Cytometry, Double Staining, Incubation

    Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P

    Journal: Cell Adhesion & Migration

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

    doi: 10.1080/19336918.2015.1103421

    Figure Lengend Snippet: Digestion of cell surface HSPGs with heparinase considerably reduces the level of surface-bound Hsp90α and Hsp90β in A-172 and HT1080 cells. Cells were incubated for 1 h at 37°C with a heparinase I/III blend, stained with anti-Hsp90α, anti-Hsp90β, and anti-heparan sulfate antibodies, and analyzed by confocal microscopy ( A ) and flow cytometry ( B ). ( A ) Representative confocal microscopy images showing the surface staining with antibodies are presented. Scale bar: 20 μm. ( B ) Representative flow cytometry histograms for control (black lines) and heparinase-treated (red lines) cells stained with Hsp90-specific antibodies, as well as for cells stained with the negative control rabbit antibody (blue lines) are presented. ( C ) Flow cytometry-based quantification of membrane-bound Hsp90α and Hsp90β expression after heparinase treatment. The data are presented as the MFI specific for Hsp90α and Hsp90β, expressed in percent. MFI of control cells was taken as 100%. Each bar represents the mean ± SD (n = 4–5). *Statistically significantly different ( P

    Article Snippet: Treatment of cells with heparinase I/III and sodium chlorate To assess the influence of surface heparan sulfate digestion on the cell surface expression of Hsp90α and Hsp90β, A-172 and HT1080 cells were washed with DMEM and incubated for 1 h at 37°C with a heparinase I/III blend from Flavobacterium heparinum (Sigma-Aldrich) diluted in DMEM-FBS (0.03 IU/ml).

    Techniques: Incubation, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Negative Control, Expressing

    N-sulfate-enriched microenvironment forms a belt around the demyelinated lesion. HS (10E4) labeling on the contra- ( A ) and ipsi- ( B–C ) lateral side to the lesion illustrates the generation of a N-sulfated microenvironment surrounding the lesion (delimited by white dashed lines) at five dpi (n = 3). No immunoreactivity was found after Heparinase I treatment ( D ) thus validating the 10E4 antibody specificity. Scale bars: 20 µm in A, B, D; 10 µm in C. CC, corpus callosum; V, ventricle.

    Journal: eLife

    Article Title: Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production

    doi: 10.7554/eLife.51735

    Figure Lengend Snippet: N-sulfate-enriched microenvironment forms a belt around the demyelinated lesion. HS (10E4) labeling on the contra- ( A ) and ipsi- ( B–C ) lateral side to the lesion illustrates the generation of a N-sulfated microenvironment surrounding the lesion (delimited by white dashed lines) at five dpi (n = 3). No immunoreactivity was found after Heparinase I treatment ( D ) thus validating the 10E4 antibody specificity. Scale bars: 20 µm in A, B, D; 10 µm in C. CC, corpus callosum; V, ventricle.

    Article Snippet: For N-sulfated motifs labeling, floating sections from PFA perfused-brain were incubated for 2 hr 30 at 37°C in buffer (100 mM Sodium Chloride, 1 mM Calcium Chloride, 50 mM Hepes 5 µg, BSA pH 7) with or without Heparinase (3.3 mU from Flavobacterium heparinum, Seikagaku Kogyo Co. # 100700, Japan) ( ) before permeabilization.

    Techniques: Labeling

    Effects of PD98059 and heparinase on SMC marker gene expression under 3-D interstitial flow. PD98059 and heparinase reverse interstitial flow-induced reductions in SM-MHC, smoothelin, and calponin expression, but further enhance α-SMA and SM22 expression in 3-D. SMCs (A) and MFBs (B) in collagen gels were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to interstitial flow (1 cmH 2 O) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: Effects of PD98059 and heparinase on SMC marker gene expression under 3-D interstitial flow. PD98059 and heparinase reverse interstitial flow-induced reductions in SM-MHC, smoothelin, and calponin expression, but further enhance α-SMA and SM22 expression in 3-D. SMCs (A) and MFBs (B) in collagen gels were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to interstitial flow (1 cmH 2 O) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Marker, Expressing, Flow Cytometry, Quantitative RT-PCR

    Cleavage of heparan sulfate glycosaminglycans (HS-GAGs) by heparinase. SMCs were grown on the plate for 2 days, and then incubated with 6.7 IU/L heparinase III (Hep) for 1 h followed by immunostaining for HS-GAGs. The surfaces of SMCs present abundant HS-GAGs (blue), which was successfully cleaved by heparinase III. Cell nuclei were stained by propidium iodide shown in red.

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: Cleavage of heparan sulfate glycosaminglycans (HS-GAGs) by heparinase. SMCs were grown on the plate for 2 days, and then incubated with 6.7 IU/L heparinase III (Hep) for 1 h followed by immunostaining for HS-GAGs. The surfaces of SMCs present abundant HS-GAGs (blue), which was successfully cleaved by heparinase III. Cell nuclei were stained by propidium iodide shown in red.

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Incubation, Immunostaining, Staining

    PD98059 and heparinase suppress both laminar flow and interstitial flow-induced ERK1/2 activation. Cells in 2-D or 3-D were pretreated with ERK1/2 inhibitor PD98059 (PD) or heparinase III (Hep) and then exposed to laminar flow or interstitial flow for 0 to 30 min. Cells were lysed and proteins were extracted for Western blotting. Gel panels were representative Western blots from three independent experiments, where similar results were found.

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: PD98059 and heparinase suppress both laminar flow and interstitial flow-induced ERK1/2 activation. Cells in 2-D or 3-D were pretreated with ERK1/2 inhibitor PD98059 (PD) or heparinase III (Hep) and then exposed to laminar flow or interstitial flow for 0 to 30 min. Cells were lysed and proteins were extracted for Western blotting. Gel panels were representative Western blots from three independent experiments, where similar results were found.

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Flow Cytometry, Activation Assay, Western Blot

    Effects of PD98059 and heparinase on SMC marker gene expression under 2-D laminar flow. PD98059 and heparinase reverse laminar flow-induced reductions in expression of SMC marker genes in 2-D. SMCs (A) and MFBs (B) were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to 8 dyn/cm 2 laminar shear stress for 15 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

    doi: 10.1371/journal.pone.0012196

    Figure Lengend Snippet: Effects of PD98059 and heparinase on SMC marker gene expression under 2-D laminar flow. PD98059 and heparinase reverse laminar flow-induced reductions in expression of SMC marker genes in 2-D. SMCs (A) and MFBs (B) were pretreated with PD98059 (PD) or heparinase III (Hepr) for 3 h, and then exposed to 8 dyn/cm 2 laminar shear stress for 15 h. Gene expression was analyzed by RT-qPCR and normalized to its own Flow without PD or Hepr treated case. All the data are presented as mean ± SEM. * P

    Article Snippet: ERK1/2 inhibition and HSPG cleavage PD98059 (Calbiochem) was used for ERK1/2 inhibition and heparinase III (IBEX Technologies, Montreal, Canada) was used for HSPG cleavage.

    Techniques: Marker, Expressing, Flow Cytometry, Quantitative RT-PCR

    NK cell–intrinsic heparanase is indispensable for efficient surveillance of MCA-induced fibrosarcoma and RAE-1–expressing lymphoma. ( A – C ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were inoculated s.c. in the hind flank with 100 μg MCA in 0.1 ml corn oil. Mice were then monitored over a 200-day period for fibrosarcoma development. Tumors were measured every week with a caliper ( n = 24–28 per group; data were pooled from 2 independent experiments). ( A ) Data were recorded as the percentage of tumor-free mice (tumors were defined as measuring > 3 mm in diameter and consistently growing). Tumor growth curves of individual ( B ) Hpse fl/fl NKp46-WT and ( C ) Hpse fl/fl NKp46-iCre mice. ( D ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected s.c. with 5 × 10 6 RMA-S-RAE-1β cells. Tumor growth was measured every 2 to 3 days with a caliper (mean ± SEM; n = 7 per group; 1 representative experiment of 2 experiments). Statistically significant differences were determined by log-rank Mantel-Cox test ( A ) and Mann-Whitney U test ( D ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: NK cell heparanase controls tumor invasion and immune surveillance

    doi: 10.1172/JCI92958

    Figure Lengend Snippet: NK cell–intrinsic heparanase is indispensable for efficient surveillance of MCA-induced fibrosarcoma and RAE-1–expressing lymphoma. ( A – C ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were inoculated s.c. in the hind flank with 100 μg MCA in 0.1 ml corn oil. Mice were then monitored over a 200-day period for fibrosarcoma development. Tumors were measured every week with a caliper ( n = 24–28 per group; data were pooled from 2 independent experiments). ( A ) Data were recorded as the percentage of tumor-free mice (tumors were defined as measuring > 3 mm in diameter and consistently growing). Tumor growth curves of individual ( B ) Hpse fl/fl NKp46-WT and ( C ) Hpse fl/fl NKp46-iCre mice. ( D ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected s.c. with 5 × 10 6 RMA-S-RAE-1β cells. Tumor growth was measured every 2 to 3 days with a caliper (mean ± SEM; n = 7 per group; 1 representative experiment of 2 experiments). Statistically significant differences were determined by log-rank Mantel-Cox test ( A ) and Mann-Whitney U test ( D ). * P

    Article Snippet: Heparanase is thus considered a highly promising target for anticancer therapy, and the first clinical trials using heparan sulfate (HS) mimetics have enrolled patients.

    Techniques: Expressing, Mouse Assay, Injection, MANN-WHITNEY

    NK cell invasion is impaired in the absence of heparanase. ( A ) The expression of CD62L and CXCR3 on TCRβ – NK1.1 + DX5 + NK cells was analyzed by flow cytometry in the indicated organs of Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice (mean ± SEM; n = 3–10). ( B ) Preactivated splenic NK cells (7.5 × 10 4 ) were seeded in the upper chamber of a Transwell insert. The number of migrating cells in response to 10% FBS and 20 ng/ml CXCL10 was assessed after 17 hours (mean ± SEM; n = 5; data were pooled from 2 independent experiments). ( C ) Heparanase enzymatic activity of isolated Hpse +/+ and Hpse –/– splenic NK cells was determined by a TR-FRET–based HS degradation assay (mean ± SD; n = 3). ( D ) Mice were injected s.c. with 100 μl Matrigel. Leukocyte infiltration into the Matrigel plugs was determined by flow cytometry after 3 days. Depicted are TCRβ – NK1.1 + NKp46 + NK, TCRβ + CD4 + , and TCRβ + CD8 + T cells, respectively (mean ± SEM; n = 9–10 mice per group; data were pooled from 3 independent experiments). ( E ) Mice were injected i.v. with 5 × 10 5 B16F10 melanoma cells. Lungs were harvested after 24 hours, and NK cell proportions and numbers were determined by flow cytometry (mean ± SEM; n = 9–12 mice per group; data were pooled from 3 independent experiments; direct comparison of NK cell proportions between B16F10 challenged NKp46-WT and NKp46-iCre mice P = 0.0118, Mann-Whitney U test, and NK cell numbers between B16F10 challenged NKp46-WT and NKp46-iCre mice: P = 0.0278, Mann-Whitney U test). ( F – H ) Mice were injected s.c. with 5 × 10 6 RMA-S-RAE-1β cells. Tumors were harvested on day 5 and analyzed by immunofluorescence (mean ± SEM; n = 4–6 per group). ( F ) Representative images of sections stained for NKp46 + NK cells (magenta) and DAPI (blue). Scale bars: 500 μm. Original magnification: ×20, tiled scan of whole tumor. ( G ) The distance of individual NK cells from the edge was calculated. ( H ) The number of NKp46 + cells per section was quantified by Imaris. Statistically significant differences were determined by Student’s t test ( C ), Mann-Whitney U test ( D and H ), or 1-way ANOVA with Tukey’s post test ( E ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: NK cell heparanase controls tumor invasion and immune surveillance

    doi: 10.1172/JCI92958

    Figure Lengend Snippet: NK cell invasion is impaired in the absence of heparanase. ( A ) The expression of CD62L and CXCR3 on TCRβ – NK1.1 + DX5 + NK cells was analyzed by flow cytometry in the indicated organs of Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice (mean ± SEM; n = 3–10). ( B ) Preactivated splenic NK cells (7.5 × 10 4 ) were seeded in the upper chamber of a Transwell insert. The number of migrating cells in response to 10% FBS and 20 ng/ml CXCL10 was assessed after 17 hours (mean ± SEM; n = 5; data were pooled from 2 independent experiments). ( C ) Heparanase enzymatic activity of isolated Hpse +/+ and Hpse –/– splenic NK cells was determined by a TR-FRET–based HS degradation assay (mean ± SD; n = 3). ( D ) Mice were injected s.c. with 100 μl Matrigel. Leukocyte infiltration into the Matrigel plugs was determined by flow cytometry after 3 days. Depicted are TCRβ – NK1.1 + NKp46 + NK, TCRβ + CD4 + , and TCRβ + CD8 + T cells, respectively (mean ± SEM; n = 9–10 mice per group; data were pooled from 3 independent experiments). ( E ) Mice were injected i.v. with 5 × 10 5 B16F10 melanoma cells. Lungs were harvested after 24 hours, and NK cell proportions and numbers were determined by flow cytometry (mean ± SEM; n = 9–12 mice per group; data were pooled from 3 independent experiments; direct comparison of NK cell proportions between B16F10 challenged NKp46-WT and NKp46-iCre mice P = 0.0118, Mann-Whitney U test, and NK cell numbers between B16F10 challenged NKp46-WT and NKp46-iCre mice: P = 0.0278, Mann-Whitney U test). ( F – H ) Mice were injected s.c. with 5 × 10 6 RMA-S-RAE-1β cells. Tumors were harvested on day 5 and analyzed by immunofluorescence (mean ± SEM; n = 4–6 per group). ( F ) Representative images of sections stained for NKp46 + NK cells (magenta) and DAPI (blue). Scale bars: 500 μm. Original magnification: ×20, tiled scan of whole tumor. ( G ) The distance of individual NK cells from the edge was calculated. ( H ) The number of NKp46 + cells per section was quantified by Imaris. Statistically significant differences were determined by Student’s t test ( C ), Mann-Whitney U test ( D and H ), or 1-way ANOVA with Tukey’s post test ( E ). * P

    Article Snippet: Heparanase is thus considered a highly promising target for anticancer therapy, and the first clinical trials using heparan sulfate (HS) mimetics have enrolled patients.

    Techniques: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Activity Assay, Isolation, Degradation Assay, Injection, MANN-WHITNEY, Immunofluorescence, Staining

    Activated NK cells express enzymatically active heparanase. ( A – E ) NK cells isolated from human donors were assayed as f-NK or a-NK cells. i-DCs were included as a control. ( A ) mRNA expression of HPSE relative to UBC was assessed by quantitative PCR (qPCR) (mean ± SD; n = 3 individual donors; 1 representative experiment of 2 experiments). ( B and C ) Heparanase protein expression was determined by intracellular staining and flow cytometry (mean ± SEM; n = 5–13 donors per group). MFI, mean fluorescence intensity. ( D ) HPSE enzymatic activity was determined by incubating 2 × 10 5 f-NK or a-NK cells with 3 H-HS for 16 hours ± 1 U heparin. Human platelet heparanase (2.5 ng) was included as a control (mean ± SEM; n = 4–11 per group; data were pooled from 2 independent experiments). ( E ) a-NK cells (2 × 10 6 ) from 2 individual donors were cultured on 35 S-ECM plates ± 2 ng/ml PMA/0.1 μM ionomycin (IO) ± 200 μg/ml PI-88. ECM degradation was measured after 20 hours (mean ± SD; n = 3 technical replicates; data are representative of 5 individual donors). ( F ) Heparanase expression was analyzed by Western blotting. FACS-purified mouse TCRβ – NK1.1 + NKp46 + DX5 + NK cells were analyzed ex vivo or after stimulation for the indicated durations by cytokines (500 U/ml IL-2, 1 ng/ml IL-12, 10 ng/ml IL-15, and 10 ng/ml IL-18) or by NK cell receptor cross-linking (α-Ly49D or α-NK1.1). ( G ) The enzymatic activity of heparanase was determined by a TR-FRET–based HS degradation assay. Splenic NK cells were isolated by negative depletion from WT mice that had been injected with 250 μg poly(I:C) 24 hours prior to the analysis or were left untreated (mean ± SD; n = 3). Statistically significant differences between the groups were determined by 1-way ANOVA with Tukey’s post test ( A , C , and D ) or unpaired Student’s t test ( G ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: NK cell heparanase controls tumor invasion and immune surveillance

    doi: 10.1172/JCI92958

    Figure Lengend Snippet: Activated NK cells express enzymatically active heparanase. ( A – E ) NK cells isolated from human donors were assayed as f-NK or a-NK cells. i-DCs were included as a control. ( A ) mRNA expression of HPSE relative to UBC was assessed by quantitative PCR (qPCR) (mean ± SD; n = 3 individual donors; 1 representative experiment of 2 experiments). ( B and C ) Heparanase protein expression was determined by intracellular staining and flow cytometry (mean ± SEM; n = 5–13 donors per group). MFI, mean fluorescence intensity. ( D ) HPSE enzymatic activity was determined by incubating 2 × 10 5 f-NK or a-NK cells with 3 H-HS for 16 hours ± 1 U heparin. Human platelet heparanase (2.5 ng) was included as a control (mean ± SEM; n = 4–11 per group; data were pooled from 2 independent experiments). ( E ) a-NK cells (2 × 10 6 ) from 2 individual donors were cultured on 35 S-ECM plates ± 2 ng/ml PMA/0.1 μM ionomycin (IO) ± 200 μg/ml PI-88. ECM degradation was measured after 20 hours (mean ± SD; n = 3 technical replicates; data are representative of 5 individual donors). ( F ) Heparanase expression was analyzed by Western blotting. FACS-purified mouse TCRβ – NK1.1 + NKp46 + DX5 + NK cells were analyzed ex vivo or after stimulation for the indicated durations by cytokines (500 U/ml IL-2, 1 ng/ml IL-12, 10 ng/ml IL-15, and 10 ng/ml IL-18) or by NK cell receptor cross-linking (α-Ly49D or α-NK1.1). ( G ) The enzymatic activity of heparanase was determined by a TR-FRET–based HS degradation assay. Splenic NK cells were isolated by negative depletion from WT mice that had been injected with 250 μg poly(I:C) 24 hours prior to the analysis or were left untreated (mean ± SD; n = 3). Statistically significant differences between the groups were determined by 1-way ANOVA with Tukey’s post test ( A , C , and D ) or unpaired Student’s t test ( G ). * P

    Article Snippet: Heparanase is thus considered a highly promising target for anticancer therapy, and the first clinical trials using heparan sulfate (HS) mimetics have enrolled patients.

    Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Fluorescence, Activity Assay, Cell Culture, Western Blot, FACS, Purification, Ex Vivo, Degradation Assay, Mouse Assay, Injection

    Heparanase-deficient NK cells display impaired control of lung metastases. ( A ) Hpse fl/fl NKp46-WT , Hpse fl/fl NKp46-iCre , Hpse WT/WT NKp46-WT (B6.WT), and Hpse WT/WT NKp46-iCre mice were injected i.v. with 1 × 10 5 RM-1 prostate carcinoma cells. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 4–16 mice per group; data were pooled from 2 independent experiments). ( B ) Hpse fl/fl NKp46-WT , Hpse fl/fl NKp46-iCre, Hpse WT/WT NKp46-WT (B6.WT), and Hpse WT/WT NKp46-iCre mice were injected i.v. with 2 × 10 5 B16F10 melanoma cells. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 6–22 mice per group; data were pooled from 3 independent experiments). ( C ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected with 2 × 10 4 E0771 cells into the mammary fat pad and treated with either 50 μg control Ig (–) or anti–asialo-GM1 (+) (NK cell depletion) on days –1, 0, 7, 14, and 23 after tumor transplantation. Tumors were removed surgically on day 12. Lungs were harvested on day 35 and macrometastases counted (mean ± SEM; n = 6–8 mice per group). ( D ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected i.v. with 5 × 10 5 B16F10 melanoma cells and treated i.p. with either PBS or 100,000 IU IL-2 on days 0, 1, 2, 3, and 4. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 10–11 mice per group; data were pooled from 2 independent experiments). ( E ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected i.v. with 5 × 10 5 B16F10 melanoma cells and treated with either 500 μg control Ig (–) or 250 μg each of anti-CTLA4 and anti–PD-1 (+) on days 0 and 3 after injection, respectively. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 5–7 mice per group). ( A – E ) Statistically significant differences between the groups were determined by 1-way ANOVA with Tukey’s post test (* P

    Journal: The Journal of Clinical Investigation

    Article Title: NK cell heparanase controls tumor invasion and immune surveillance

    doi: 10.1172/JCI92958

    Figure Lengend Snippet: Heparanase-deficient NK cells display impaired control of lung metastases. ( A ) Hpse fl/fl NKp46-WT , Hpse fl/fl NKp46-iCre , Hpse WT/WT NKp46-WT (B6.WT), and Hpse WT/WT NKp46-iCre mice were injected i.v. with 1 × 10 5 RM-1 prostate carcinoma cells. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 4–16 mice per group; data were pooled from 2 independent experiments). ( B ) Hpse fl/fl NKp46-WT , Hpse fl/fl NKp46-iCre, Hpse WT/WT NKp46-WT (B6.WT), and Hpse WT/WT NKp46-iCre mice were injected i.v. with 2 × 10 5 B16F10 melanoma cells. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 6–22 mice per group; data were pooled from 3 independent experiments). ( C ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected with 2 × 10 4 E0771 cells into the mammary fat pad and treated with either 50 μg control Ig (–) or anti–asialo-GM1 (+) (NK cell depletion) on days –1, 0, 7, 14, and 23 after tumor transplantation. Tumors were removed surgically on day 12. Lungs were harvested on day 35 and macrometastases counted (mean ± SEM; n = 6–8 mice per group). ( D ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected i.v. with 5 × 10 5 B16F10 melanoma cells and treated i.p. with either PBS or 100,000 IU IL-2 on days 0, 1, 2, 3, and 4. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 10–11 mice per group; data were pooled from 2 independent experiments). ( E ) Hpse fl/fl NKp46-WT and Hpse fl/fl NKp46-iCre mice were injected i.v. with 5 × 10 5 B16F10 melanoma cells and treated with either 500 μg control Ig (–) or 250 μg each of anti-CTLA4 and anti–PD-1 (+) on days 0 and 3 after injection, respectively. Lungs were harvested on day 14 and macrometastases counted (mean ± SEM; n = 5–7 mice per group). ( A – E ) Statistically significant differences between the groups were determined by 1-way ANOVA with Tukey’s post test (* P

    Article Snippet: Heparanase is thus considered a highly promising target for anticancer therapy, and the first clinical trials using heparan sulfate (HS) mimetics have enrolled patients.

    Techniques: Mouse Assay, Injection, Transplantation Assay

    NK cell proliferation and function are unchanged by loss of heparanase. ( A and B ) Purified BM NK cells from Hpse fl/fl NKp46-WT or Hpse fl/fl NKp46-iCre mice were labeled with CTV and cultured for 3 days in IL-15 as indicated (mean ± SD; n = 2 biological replicates). ( A ) Apoptosis was determined by annexin V and propidium iodide staining. ( B ) Proliferation was assessed by CTV dilution. ( C ) Purified splenic CFSE-labeled NK cells (2 × 10 5 ) were injected i.v. into B6. Rag2 –/– Il2rg –/– mice. After 3 days, the proliferation of CD45 + TCRβ – NK1.1 + DX5 + NK cells in the indicated organs was determined by flow cytometry. Flow cytometric plot shows a representative proliferation profile. Data in the bar graph were pooled from 2 independent experiments (mean ± SEM; n = 8 per group). ( D ) The cytotoxicity of freshly isolated splenocytes or IL-2–activated NK cells (1,000 U/ml for 5 days) against YAC-1 and B16F10 target cells was tested at the indicated E/T ratios after 4 hours (mean ± SD; n = 3 biological replicates; 1 representative experiment of 2 experiments). ( E ) Splenocytes (5 × 10 6 ) were stimulated for 4 hours with 1 ng/ml IL-12, 100 ng/ml IL-15, and 10 ng/ml IL-18, and the expression of CD107a was assessed on TCRβ – NK1.1 + DX5 + NK cells (mean ± SD; n = 4 mice per group). ( F ) Lung cells were stimulated for 4 hours in 1 ng/ml IL-12, 100 ng/ml IL-15, and 10 ng/ml IL-18, and the production of IFN-γ was measured by intracellular staining (mean ± SEM; n = 10; data were pooled from 3 independent experiments). ( G ) Purified splenic NK cells were stimulated in 50 ng/ml IL-15, 100 ng/ml IL-21, 1 ng/ml IL-12, 10 ng/ml IL-18, or anti-NK1.1 precoated wells. The release of IFN-γ was measured after 24 hours by CBA (mean ± SD; n = 2 biological replicates; 1 representative experiment of 2 experiments).

    Journal: The Journal of Clinical Investigation

    Article Title: NK cell heparanase controls tumor invasion and immune surveillance

    doi: 10.1172/JCI92958

    Figure Lengend Snippet: NK cell proliferation and function are unchanged by loss of heparanase. ( A and B ) Purified BM NK cells from Hpse fl/fl NKp46-WT or Hpse fl/fl NKp46-iCre mice were labeled with CTV and cultured for 3 days in IL-15 as indicated (mean ± SD; n = 2 biological replicates). ( A ) Apoptosis was determined by annexin V and propidium iodide staining. ( B ) Proliferation was assessed by CTV dilution. ( C ) Purified splenic CFSE-labeled NK cells (2 × 10 5 ) were injected i.v. into B6. Rag2 –/– Il2rg –/– mice. After 3 days, the proliferation of CD45 + TCRβ – NK1.1 + DX5 + NK cells in the indicated organs was determined by flow cytometry. Flow cytometric plot shows a representative proliferation profile. Data in the bar graph were pooled from 2 independent experiments (mean ± SEM; n = 8 per group). ( D ) The cytotoxicity of freshly isolated splenocytes or IL-2–activated NK cells (1,000 U/ml for 5 days) against YAC-1 and B16F10 target cells was tested at the indicated E/T ratios after 4 hours (mean ± SD; n = 3 biological replicates; 1 representative experiment of 2 experiments). ( E ) Splenocytes (5 × 10 6 ) were stimulated for 4 hours with 1 ng/ml IL-12, 100 ng/ml IL-15, and 10 ng/ml IL-18, and the expression of CD107a was assessed on TCRβ – NK1.1 + DX5 + NK cells (mean ± SD; n = 4 mice per group). ( F ) Lung cells were stimulated for 4 hours in 1 ng/ml IL-12, 100 ng/ml IL-15, and 10 ng/ml IL-18, and the production of IFN-γ was measured by intracellular staining (mean ± SEM; n = 10; data were pooled from 3 independent experiments). ( G ) Purified splenic NK cells were stimulated in 50 ng/ml IL-15, 100 ng/ml IL-21, 1 ng/ml IL-12, 10 ng/ml IL-18, or anti-NK1.1 precoated wells. The release of IFN-γ was measured after 24 hours by CBA (mean ± SD; n = 2 biological replicates; 1 representative experiment of 2 experiments).

    Article Snippet: Heparanase is thus considered a highly promising target for anticancer therapy, and the first clinical trials using heparan sulfate (HS) mimetics have enrolled patients.

    Techniques: Purification, Mouse Assay, Labeling, Cell Culture, Staining, Injection, Flow Cytometry, Cytometry, Isolation, Expressing, Crocin Bleaching Assay

    GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K d s were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125 I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with heparinase III (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125 I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.

    Journal: The Journal of Cell Biology

    Article Title: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

    doi: 10.1083/jcb.201009136

    Figure Lengend Snippet: GDNF, NRTN, and ARTN bind directly to syndecan-3 with high affinity. (A) Syndecan-3 (S3) binding to GFLs, immobilized on the cuvette, was followed by SPR. The K d s were determined from dynamics as well as equilibrium data (insets), and their values are indicated in the graphs. Error bars show SEM from three to four experiments for each condition. K on is the association rate. arcsec, arcsecond. conc, concentration. (B) GDNF interacts directly with syndecan-3 in the rat glioma C6 cell line. Chemical cross-linking of 125 I-GDNF to C6 cells was followed by immunoprecipitation (IP) with antisyndecan-3 antibodies in untreated cells (UNTR), cells pretreated with heparinase III (H’ase III), and cells pretreated with phosphatidylinositol-specific PLC (PI-PLC). Alternatively, a 150-fold molar excess of unlabeled (cold) GDNF and HB-GAM or 1 µg/ml heparin was added simultaneously with 125 I-GDNF. In the negative control, no syndecan-3 antibodies were added (No AB). The high molecular mass band in the top of the gel corresponds to the GDNF–syndecan-3 complex. Equal loading of the proteins was confirmed by silver staining of the gel (not depicted). (C) Western blotting (WB) of proteins extracted from dissociated P9 mouse brain and kidney tissues. Lysates from ECM and membrane-associated fractions were separated by SDS-PAGE, and Western blots were probed with anti-GDNF (top) or anti–HB-GAM antibodies (bottom). The bands corresponding to GDNF and HB-GAM are marked with asterisks. Molecular mass markers are shown on the right. (D) GDNF-induced syndecan-3 oligomerization visualized by FRET. FRET channel images of human embryonic kidney cells transiently transfected with syndecan-3 fusion constructs with YFP or CFP and stimulated with 100 ng/ml GDNF. Images were taken after GDNF stimulation at varying time points. FRET-corrected images were displayed in pseudocolors (red areas indicate high values of FRET, and blue areas indicate low values of FRET). Insets highlight a strong FRET signal at the plasma membrane, where syndecan-3 is targeted. Bar, 10 µM.

    Article Snippet: Heparinase III was obtained from Seikagaku Corp. (Seikagaku’s trademark for this product is Heparitinase I).

    Techniques: Binding Assay, SPR Assay, Concentration Assay, Immunoprecipitation, Planar Chromatography, Negative Control, Silver Staining, Western Blot, SDS Page, Transfection, Construct

    GDNF, NRTN, and ARTN induce SHEP cell adherence and SFK activation in an HSPG-dependent manner. (A) RT-PCR of the cDNA from SHEP cells with syndecan-3–specific primers. The resulting 140-bp band represents a syndecan-3 mRNA-derived PCR product (lane 2). (lane 1) Molecular weight markers with sizes in bp. (B) Crystal violet staining of SHEP cells plated on immobilized GDNF, ARTN, NRTN, and PSPN with or without heparinase III (H’aseIII) pretreatment. (C) Quantification of attached SHEP cells on immobilized GFLs. Some cells were treated with heparinase III. Quantification also includes controls with cell attachment on BSA and attachment of cells treated with heparinase III on tissue culture plates. (D) Quantification of SHEP cell adherence to immobilized GDNF and ΔN-GDNF. Cells were plated on GDNF untreated, or they were pretreated with PI-PLC, chondroitinase ABC (Ch’ase ABC), NCAM function-blocking antibodies (NCAM BLK), or GDNF function-blocking antibodies (GDNF BLK). (C and D) Error bars show SEM from three independent assays. (E) Western blot (WB) for activated SFK in lysates from SHEP cells plated on GDNF, ΔN-GDNF, or BSA. In control experiments, cells plated on GDNF were treated with heparinase III (HIII) or PI-PLC. Western blots were probed with anti-pY 418 Src (top) or anti-Src (bottom) antibodies. (F) SHEP cell adherence and spreading on immobilized GDNF in the absence or presence of 2 µM SFK inhibitor SU6656. Cell spreading on immobilized GDNF was impaired in the presence of the inhibitor, whereas adherence of SHEP cells was not significantly affected by SU6656. (G) Adherence and spreading of SHEP cells infected with adenovirus expressing GFP (AdGFP) or dominant-negative Src (AdDN-Src/GFP) on immobilized GDNF. Transduction of cells with AdDN-Src/GFP resulted in SHEP’s failure to spread on immobilized GDNF. GFP-expressing adenovirus did not affect SHEP cell spreading on the GDNF matrix. Untreated, UNTR. Bars, 100 µM.

    Journal: The Journal of Cell Biology

    Article Title: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

    doi: 10.1083/jcb.201009136

    Figure Lengend Snippet: GDNF, NRTN, and ARTN induce SHEP cell adherence and SFK activation in an HSPG-dependent manner. (A) RT-PCR of the cDNA from SHEP cells with syndecan-3–specific primers. The resulting 140-bp band represents a syndecan-3 mRNA-derived PCR product (lane 2). (lane 1) Molecular weight markers with sizes in bp. (B) Crystal violet staining of SHEP cells plated on immobilized GDNF, ARTN, NRTN, and PSPN with or without heparinase III (H’aseIII) pretreatment. (C) Quantification of attached SHEP cells on immobilized GFLs. Some cells were treated with heparinase III. Quantification also includes controls with cell attachment on BSA and attachment of cells treated with heparinase III on tissue culture plates. (D) Quantification of SHEP cell adherence to immobilized GDNF and ΔN-GDNF. Cells were plated on GDNF untreated, or they were pretreated with PI-PLC, chondroitinase ABC (Ch’ase ABC), NCAM function-blocking antibodies (NCAM BLK), or GDNF function-blocking antibodies (GDNF BLK). (C and D) Error bars show SEM from three independent assays. (E) Western blot (WB) for activated SFK in lysates from SHEP cells plated on GDNF, ΔN-GDNF, or BSA. In control experiments, cells plated on GDNF were treated with heparinase III (HIII) or PI-PLC. Western blots were probed with anti-pY 418 Src (top) or anti-Src (bottom) antibodies. (F) SHEP cell adherence and spreading on immobilized GDNF in the absence or presence of 2 µM SFK inhibitor SU6656. Cell spreading on immobilized GDNF was impaired in the presence of the inhibitor, whereas adherence of SHEP cells was not significantly affected by SU6656. (G) Adherence and spreading of SHEP cells infected with adenovirus expressing GFP (AdGFP) or dominant-negative Src (AdDN-Src/GFP) on immobilized GDNF. Transduction of cells with AdDN-Src/GFP resulted in SHEP’s failure to spread on immobilized GDNF. GFP-expressing adenovirus did not affect SHEP cell spreading on the GDNF matrix. Untreated, UNTR. Bars, 100 µM.

    Article Snippet: Heparinase III was obtained from Seikagaku Corp. (Seikagaku’s trademark for this product is Heparitinase I).

    Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Molecular Weight, Staining, Cell Attachment Assay, Planar Chromatography, Blocking Assay, Western Blot, Infection, Expressing, Dominant Negative Mutation, Transduction

    Immobilized GDNF induces neurite outgrowth in rat embryonic hippocampal neurons. (A) Neurite outgrowth in E17 rat hippocampal neurons on immobilized GDNF. Neurons are stained with tubulin-βIII antibodies. Bar, 20 µM. (B) Neurite outgrowth in hippocampal neurons plated on BSA. Neurons are stained with tubulin-βIII antibodies. (C) Quantification of neurite outgrowth on immobilized GDNF, ΔN-GDNF, PSPN, and BSA. Neurons plated on GDNF were untreated (UNTR) or preincubated with heparinase III (H’aseIII), PI-PLC, or 2 µM SFK inhibitor SU6656. As a control, soluble GDNF was added to neurons grown on BSA. Error bars show SEM from three to five independent experiments (*, P

    Journal: The Journal of Cell Biology

    Article Title: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

    doi: 10.1083/jcb.201009136

    Figure Lengend Snippet: Immobilized GDNF induces neurite outgrowth in rat embryonic hippocampal neurons. (A) Neurite outgrowth in E17 rat hippocampal neurons on immobilized GDNF. Neurons are stained with tubulin-βIII antibodies. Bar, 20 µM. (B) Neurite outgrowth in hippocampal neurons plated on BSA. Neurons are stained with tubulin-βIII antibodies. (C) Quantification of neurite outgrowth on immobilized GDNF, ΔN-GDNF, PSPN, and BSA. Neurons plated on GDNF were untreated (UNTR) or preincubated with heparinase III (H’aseIII), PI-PLC, or 2 µM SFK inhibitor SU6656. As a control, soluble GDNF was added to neurons grown on BSA. Error bars show SEM from three to five independent experiments (*, P

    Article Snippet: Heparinase III was obtained from Seikagaku Corp. (Seikagaku’s trademark for this product is Heparitinase I).

    Techniques: Staining, Planar Chromatography

    Both HS and protein components of SCM are required for boundary formation Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); heparinase treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75 NTR (green). The number of cells mingling with astrocytes was counted across a 300 μm line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate ± SEM. Scale bar 50 μm. ** p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation

    doi: 10.1523/JNEUROSCI.6340-11.2012

    Figure Lengend Snippet: Both HS and protein components of SCM are required for boundary formation Confrontation assays of OECs and astrocytes were carried out in the presence of; normal medium/untreated (A); SCM (B); heparinase treated SCM (C); trypsin treated SCM (D); 1:1 combination of heparinase treated and trypsin treated SCM (E). After 2 days of treatment, cells were fixed and stained for GFAP (red) and p75 NTR (green). The number of cells mingling with astrocytes was counted across a 300 μm line (F). Heparinase or trypsin treated SCM did not induce boundary formation when added to assays individually, however, when combined, a boundary formed, suggesting that both an HS and protein component are required for activity. Error bars indicate ± SEM. Scale bar 50 μm. ** p

    Article Snippet: HS was digested by the addition of 10 mU each of heparinase I (EC 4.2.2.7), II (EC number not assigned) and III (EC 4.2.2.8) (Ibex Technologies, Montreal, Canada) to 4 ml SCM, followed by incubation at 37°C for 6 h. A further 10 mU of each heparinase enzyme was added to SCM and the reaction incubated overnight at 37°C.

    Techniques: Staining, Activity Assay

    SAX-HPLC analysis of fluorescently labelled HS disaccharides purified from OCM and SCM indicate that SCs and OECs secrete distinct HS structures HS purified from OCM (A) and SCM (B) was digested with heparinase enzymes to generate HS disaccharides, which were fluorescently labelled with BODIPY and separated by SAX-HPLC over a 45 minute, 0-1.5 M NaCl gradient, (A and B, black lines). HS disaccharide standards were separated over the same gradient as a reference guide (A and B, dashed lines). Relative abundances of the eight HS standard disaccharides in each conditioned medium sample were calculated as a percentage of total HS (C). A summary table detailing the composition of OCM HS and SCM HS is also presented (D). SCM HS was found to be more highly sulfated than OCM HS, with an average of 1.02 sulfates per disaccharide compared to 0.75 sulfates per disaccharide in OCM HS (D). SCM HS contained a higher proportion of di- and tri-sulfated disaccharides (disaccharides 4, 5, 8 and 6) and the singly 6-O-sulfated disaccharide (disaccharide 2) compared to OCM HS (C), which contained a higher proportion of the unsulfated disaccharide (disaccharide 1) (C, D). (UA) uronic acid, (GlcN) glucosamine, (NAc) N-acetyl, (NS) N-sulfate, (6OS) 6-O-sulfate, (2OS) 2-O-sulfate.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation

    doi: 10.1523/JNEUROSCI.6340-11.2012

    Figure Lengend Snippet: SAX-HPLC analysis of fluorescently labelled HS disaccharides purified from OCM and SCM indicate that SCs and OECs secrete distinct HS structures HS purified from OCM (A) and SCM (B) was digested with heparinase enzymes to generate HS disaccharides, which were fluorescently labelled with BODIPY and separated by SAX-HPLC over a 45 minute, 0-1.5 M NaCl gradient, (A and B, black lines). HS disaccharide standards were separated over the same gradient as a reference guide (A and B, dashed lines). Relative abundances of the eight HS standard disaccharides in each conditioned medium sample were calculated as a percentage of total HS (C). A summary table detailing the composition of OCM HS and SCM HS is also presented (D). SCM HS was found to be more highly sulfated than OCM HS, with an average of 1.02 sulfates per disaccharide compared to 0.75 sulfates per disaccharide in OCM HS (D). SCM HS contained a higher proportion of di- and tri-sulfated disaccharides (disaccharides 4, 5, 8 and 6) and the singly 6-O-sulfated disaccharide (disaccharide 2) compared to OCM HS (C), which contained a higher proportion of the unsulfated disaccharide (disaccharide 1) (C, D). (UA) uronic acid, (GlcN) glucosamine, (NAc) N-acetyl, (NS) N-sulfate, (6OS) 6-O-sulfate, (2OS) 2-O-sulfate.

    Article Snippet: HS was digested by the addition of 10 mU each of heparinase I (EC 4.2.2.7), II (EC number not assigned) and III (EC 4.2.2.8) (Ibex Technologies, Montreal, Canada) to 4 ml SCM, followed by incubation at 37°C for 6 h. A further 10 mU of each heparinase enzyme was added to SCM and the reaction incubated overnight at 37°C.

    Techniques: High Performance Liquid Chromatography, Purification

    ( A ) Overview of the heparanase–GlcA complex structure. The 8 and 50 kDa subunits are depicted with yellow and purple carbons, respectively, while the arrows indicate the location of the GS3 peptide. The TIM-barrel and β-sandwich domains of heparanase are contoured with red and green lines, respectively. GlcA is represented as spheres with white carbons. ( B ) Close-up view of the catalytic site of heparanase (white cartoons and carbons). The two catalytic residues are colored in yellow and GlcA in green.

    Journal: Glycobiology

    Article Title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

    doi: 10.1093/glycob/cww003

    Figure Lengend Snippet: ( A ) Overview of the heparanase–GlcA complex structure. The 8 and 50 kDa subunits are depicted with yellow and purple carbons, respectively, while the arrows indicate the location of the GS3 peptide. The TIM-barrel and β-sandwich domains of heparanase are contoured with red and green lines, respectively. GlcA is represented as spheres with white carbons. ( B ) Close-up view of the catalytic site of heparanase (white cartoons and carbons). The two catalytic residues are colored in yellow and GlcA in green.

    Article Snippet: Thus far, drug development campaigns aimed at identifying heparanase inhibitors yielded two main classes of non-peptide and non-nucleic acid compounds, i.e., small molecules and HS mimetics ( ; ).

    Techniques:

    Heparanase-compound 1 ( A ) and heparanase-fondaparinux ( B ) complex structures obtained from the IFD procedure. This figure is available in black and white in print and in color at Glycobiology online.

    Journal: Glycobiology

    Article Title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

    doi: 10.1093/glycob/cww003

    Figure Lengend Snippet: Heparanase-compound 1 ( A ) and heparanase-fondaparinux ( B ) complex structures obtained from the IFD procedure. This figure is available in black and white in print and in color at Glycobiology online.

    Article Snippet: Thus far, drug development campaigns aimed at identifying heparanase inhibitors yielded two main classes of non-peptide and non-nucleic acid compounds, i.e., small molecules and HS mimetics ( ; ).

    Techniques:

    Schematic representation of the hypotheti c interaction between a single roneparstat chain (spheres) and two heparanase molecules (light gray). The spheres corresponding to glycol-split fragments are colored in magenta, while HBD-1, HBD-2 and the two catalytic residues are highlighted in orange, blue and red, respectively.

    Journal: Glycobiology

    Article Title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

    doi: 10.1093/glycob/cww003

    Figure Lengend Snippet: Schematic representation of the hypotheti c interaction between a single roneparstat chain (spheres) and two heparanase molecules (light gray). The spheres corresponding to glycol-split fragments are colored in magenta, while HBD-1, HBD-2 and the two catalytic residues are highlighted in orange, blue and red, respectively.

    Article Snippet: Thus far, drug development campaigns aimed at identifying heparanase inhibitors yielded two main classes of non-peptide and non-nucleic acid compounds, i.e., small molecules and HS mimetics ( ; ).

    Techniques:

    Heparanase relative activity, measured as amount of substrate cleaved at 3 h, vs. fondaparinux initial concentration (log scale). Hashed line: fitted Michaelis–Menten function. Continuous line: fitted Hill function with n H = 2 (see text). Vertical bars represent standard deviation of single measurements ( n = 4).

    Journal: Glycobiology

    Article Title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

    doi: 10.1093/glycob/cww003

    Figure Lengend Snippet: Heparanase relative activity, measured as amount of substrate cleaved at 3 h, vs. fondaparinux initial concentration (log scale). Hashed line: fitted Michaelis–Menten function. Continuous line: fitted Hill function with n H = 2 (see text). Vertical bars represent standard deviation of single measurements ( n = 4).

    Article Snippet: Thus far, drug development campaigns aimed at identifying heparanase inhibitors yielded two main classes of non-peptide and non-nucleic acid compounds, i.e., small molecules and HS mimetics ( ; ).

    Techniques: Activity Assay, Concentration Assay, Standard Deviation

    Chemical structure of heparanase inhibitor roneparstat. Given the heterogeneous saccharide composition, only a representative sequence is depicted, with m ranging from 1 to 5. Adapted from Casu et al. (2008 ).

    Journal: Glycobiology

    Article Title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

    doi: 10.1093/glycob/cww003

    Figure Lengend Snippet: Chemical structure of heparanase inhibitor roneparstat. Given the heterogeneous saccharide composition, only a representative sequence is depicted, with m ranging from 1 to 5. Adapted from Casu et al. (2008 ).

    Article Snippet: Thus far, drug development campaigns aimed at identifying heparanase inhibitors yielded two main classes of non-peptide and non-nucleic acid compounds, i.e., small molecules and HS mimetics ( ; ).

    Techniques: Sequencing

    ( A ) Superposition of human heparanase crystal structure (pink ribbons, PDB code: 5E9C) and the homology model of GS3 construct (blue ribbons). ( B and C ) Close-up views of the ligand-binding site of heparanase crystal in complex with ligand dp4 (ΔHexA2S-GlcNS6S-IdoA-GlcNS6S, pink carbons) and heparanase GS3 construct in complex with fondaparinux (blue carbons). (B) depicts the residues delimiting the catalytic sites, (C) shows the ligand molecules dp4 and roneparstat.

    Journal: Glycobiology

    Article Title: Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

    doi: 10.1093/glycob/cww003

    Figure Lengend Snippet: ( A ) Superposition of human heparanase crystal structure (pink ribbons, PDB code: 5E9C) and the homology model of GS3 construct (blue ribbons). ( B and C ) Close-up views of the ligand-binding site of heparanase crystal in complex with ligand dp4 (ΔHexA2S-GlcNS6S-IdoA-GlcNS6S, pink carbons) and heparanase GS3 construct in complex with fondaparinux (blue carbons). (B) depicts the residues delimiting the catalytic sites, (C) shows the ligand molecules dp4 and roneparstat.

    Article Snippet: Thus far, drug development campaigns aimed at identifying heparanase inhibitors yielded two main classes of non-peptide and non-nucleic acid compounds, i.e., small molecules and HS mimetics ( ; ).

    Techniques: Construct, Ligand Binding Assay

    Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Journal: Scientific Reports

    Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

    doi: 10.1038/s41598-018-27566-z

    Figure Lengend Snippet: Leukocyte adhesion as a function of treatments and varying extensional stresses. ( a ) Mean number of adherent cells per ROI in response to LFA-1 activating antibody mAb24, LFA-1 blockade antibody AF1730, pertussis toxin, and Heparinase III. Of these, only AF1730 had a significant effect. Each point is the mean value for one participant. N = 5 donors. Boxes indicate mean and standard error. Isotype controls and untreated channels are averaged into ‘baseline’ for display clarity purposes – statistics were calculated for antibodies vs. paired isotype. ( b ) Spatial linear model fit for the contribution of AF1730 to adhesion probability for wall adherent leukocytes in straight channels. The strongest effect of AF1730 blockade of LFA-1 is observed immediately after the expansion.

    Article Snippet: Heparinase III (R & D Systems) was used at 40 micrograms per milliliter.

    Techniques:

    Pancreas inflammation, cytokine induction and signaling pathways evoked by cerulein are attenuated by heparanase inhibitor. WT and Hpa-Tg mice were injected with saline (left panels) or cerulein in the absence (middle panels) or presence (right panels) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for neutrophils infiltration ( A ), p65 ( D ) and phospho-STAT3 ( F ). Immunofluorescent staining of corresponding sections for TNFα (green) is shown in ( C ) along with nuclear counterstaining (blue). Shown are representative photomicrographs at x10 ( A ) and x40 ( C,D,F ) original magnification. Total RNA was extracted from corresponding pancreas tissues and subjected to real-time PCR analyses applying primers specific for TNFα ( B ) and IL-6 ( E ). Relative gene expression (fold-increase) is shown graphically in relation to the levels in control pancreas set arbitrarily to a value of 1. *p

    Journal: Scientific Reports

    Article Title: The Role of Heparanase in the Pathogenesis of Acute Pancreatitis: A Potential Therapeutic Target

    doi: 10.1038/s41598-017-00715-6

    Figure Lengend Snippet: Pancreas inflammation, cytokine induction and signaling pathways evoked by cerulein are attenuated by heparanase inhibitor. WT and Hpa-Tg mice were injected with saline (left panels) or cerulein in the absence (middle panels) or presence (right panels) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for neutrophils infiltration ( A ), p65 ( D ) and phospho-STAT3 ( F ). Immunofluorescent staining of corresponding sections for TNFα (green) is shown in ( C ) along with nuclear counterstaining (blue). Shown are representative photomicrographs at x10 ( A ) and x40 ( C,D,F ) original magnification. Total RNA was extracted from corresponding pancreas tissues and subjected to real-time PCR analyses applying primers specific for TNFα ( B ) and IL-6 ( E ). Relative gene expression (fold-increase) is shown graphically in relation to the levels in control pancreas set arbitrarily to a value of 1. *p

    Article Snippet: Slides were incubated with 10% normal goat serum (NGS) in phosphate buffered saline (PBS) for 60 min to block nonspecific binding and incubated overnight at 4 °C with antibodies directed against LC-3 (Cell signaling #2775), cathepsin L (1:200, R & D systems, #AF1515), heparanase (1:150, #733 raised against the N terminus of the 50 kDa heparanase subunit) , p65 NFκB subunit (1:100, Santa Cruz Biotechnology, sc-372), and pSTAT3 (1:100, Santa Cruz Biotechnology, sc-8059) diluted in blocking solution.

    Techniques: Mouse Assay, Injection, Formalin-fixed Paraffin-Embedded, Staining, Real-time Polymerase Chain Reaction, Expressing

    Cerulein induces the expression of cathepsin-L in a heparanase-dependent manner. ( A ) Real-time PCR. WT and Hpa-Tg mice were injected with saline or cerulein in the absence or presence of PG545 pretreatment. Total RNA was extracted from pancreas tissues 24 hours thereafter and subjected to real-time PCR applying primers specific for cathepsin L. Relative gene expression (fold-increase) is shown graphically in relation to the levels in control pancreas set arbitrarily to a value of 1. ( B ) Immunoblotting. Proteins were extracted from corresponding pancreas tissues and lysate samples were subjected to immunoblotting applying anti-cathepsin L (upper panel) and anti-actin (lower panel) antibodies. ( C ) Immunostaining. Corresponding paraffin embedded pancreatic tissue sections were subjected to immunostaining applying anti- cathepsin-L antibody. Note a striking increase in cathepsin L expression by cerulein which is restored by the heparanase inhibitor PG545. ( D ) Colocalization of cathepsin L and heparanase. Paraffin embedded, pancreatic tissue sections of cerulein treated Hpa-Tg mice were subjected to double immunofluorescence staining using antibodies against cathepsin L (green) and heparanase (red). Merged image is shown together with nuclei counterstaining (blue). Co-localization appears in yellow. Shown are representative photomicrographs at original magnification x40.

    Journal: Scientific Reports

    Article Title: The Role of Heparanase in the Pathogenesis of Acute Pancreatitis: A Potential Therapeutic Target

    doi: 10.1038/s41598-017-00715-6

    Figure Lengend Snippet: Cerulein induces the expression of cathepsin-L in a heparanase-dependent manner. ( A ) Real-time PCR. WT and Hpa-Tg mice were injected with saline or cerulein in the absence or presence of PG545 pretreatment. Total RNA was extracted from pancreas tissues 24 hours thereafter and subjected to real-time PCR applying primers specific for cathepsin L. Relative gene expression (fold-increase) is shown graphically in relation to the levels in control pancreas set arbitrarily to a value of 1. ( B ) Immunoblotting. Proteins were extracted from corresponding pancreas tissues and lysate samples were subjected to immunoblotting applying anti-cathepsin L (upper panel) and anti-actin (lower panel) antibodies. ( C ) Immunostaining. Corresponding paraffin embedded pancreatic tissue sections were subjected to immunostaining applying anti- cathepsin-L antibody. Note a striking increase in cathepsin L expression by cerulein which is restored by the heparanase inhibitor PG545. ( D ) Colocalization of cathepsin L and heparanase. Paraffin embedded, pancreatic tissue sections of cerulein treated Hpa-Tg mice were subjected to double immunofluorescence staining using antibodies against cathepsin L (green) and heparanase (red). Merged image is shown together with nuclei counterstaining (blue). Co-localization appears in yellow. Shown are representative photomicrographs at original magnification x40.

    Article Snippet: Slides were incubated with 10% normal goat serum (NGS) in phosphate buffered saline (PBS) for 60 min to block nonspecific binding and incubated overnight at 4 °C with antibodies directed against LC-3 (Cell signaling #2775), cathepsin L (1:200, R & D systems, #AF1515), heparanase (1:150, #733 raised against the N terminus of the 50 kDa heparanase subunit) , p65 NFκB subunit (1:100, Santa Cruz Biotechnology, sc-372), and pSTAT3 (1:100, Santa Cruz Biotechnology, sc-8059) diluted in blocking solution.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Injection, Immunostaining, Double Immunofluorescence Staining

    ( A ) Cerulein treatment induces heparanase expression. Pancreas tissue was harvested from control mice (WT) or mice treated with cerulein in the absence (wt+cer) or presence of PG545 (wt+cer+PG). Total RNA was extracted and subjected to quantitative real-time PCR applying heparanase primers. Relative heparanase gene expression (fold-increase) is shown graphically in relation to heparanase levels in control pancreas set arbitrarily to a value of 1. Heparanase mRNA levels were normalized to actin mRNA (number of mice in each group = 5); *p

    Journal: Scientific Reports

    Article Title: The Role of Heparanase in the Pathogenesis of Acute Pancreatitis: A Potential Therapeutic Target

    doi: 10.1038/s41598-017-00715-6

    Figure Lengend Snippet: ( A ) Cerulein treatment induces heparanase expression. Pancreas tissue was harvested from control mice (WT) or mice treated with cerulein in the absence (wt+cer) or presence of PG545 (wt+cer+PG). Total RNA was extracted and subjected to quantitative real-time PCR applying heparanase primers. Relative heparanase gene expression (fold-increase) is shown graphically in relation to heparanase levels in control pancreas set arbitrarily to a value of 1. Heparanase mRNA levels were normalized to actin mRNA (number of mice in each group = 5); *p

    Article Snippet: Slides were incubated with 10% normal goat serum (NGS) in phosphate buffered saline (PBS) for 60 min to block nonspecific binding and incubated overnight at 4 °C with antibodies directed against LC-3 (Cell signaling #2775), cathepsin L (1:200, R & D systems, #AF1515), heparanase (1:150, #733 raised against the N terminus of the 50 kDa heparanase subunit) , p65 NFκB subunit (1:100, Santa Cruz Biotechnology, sc-372), and pSTAT3 (1:100, Santa Cruz Biotechnology, sc-8059) diluted in blocking solution.

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

    Histological analyses. ( A ) H E staining. WT and Hpa-Tg mice were injected with saline (left panels) or cerulein in the absence (middle panels) or presence (right panels) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for H E ( A ). Shown are representative photomicrographs at 10x original magnification. Corresponding pancreas tissues were processed for electron microscopy as described under ‘Materials and Methods’ ( B ). Note, increased autophagy in pancreatic acinar cells of Hpa-Tg vs. WT mice; Autophagy is further enhanced by cerulein, but restored by PG545 (cerulein+PG). Induction of AP resulted in dilatation of RER, mitochondrial swelling and large autophagosomes. Pancreatic tissue from mice treated with PG545 exhibited nearly normal ultrastructural appearance. Shown are representative micrographs at x8000, x10,000 and x12,500 original magnification. ( C ) LC-3 immunostaining. Hpa-Tg mice were injected with saline (left) or cerulein in the absence (middle) or presence (right) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for LC-3 (marker of autophagy). Shown are representative photomicrographs at original magnification of x40. Note that LC-3 staining resembles the patchy pattern of heparanase staining (Fig. 1D,E ).

    Journal: Scientific Reports

    Article Title: The Role of Heparanase in the Pathogenesis of Acute Pancreatitis: A Potential Therapeutic Target

    doi: 10.1038/s41598-017-00715-6

    Figure Lengend Snippet: Histological analyses. ( A ) H E staining. WT and Hpa-Tg mice were injected with saline (left panels) or cerulein in the absence (middle panels) or presence (right panels) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for H E ( A ). Shown are representative photomicrographs at 10x original magnification. Corresponding pancreas tissues were processed for electron microscopy as described under ‘Materials and Methods’ ( B ). Note, increased autophagy in pancreatic acinar cells of Hpa-Tg vs. WT mice; Autophagy is further enhanced by cerulein, but restored by PG545 (cerulein+PG). Induction of AP resulted in dilatation of RER, mitochondrial swelling and large autophagosomes. Pancreatic tissue from mice treated with PG545 exhibited nearly normal ultrastructural appearance. Shown are representative micrographs at x8000, x10,000 and x12,500 original magnification. ( C ) LC-3 immunostaining. Hpa-Tg mice were injected with saline (left) or cerulein in the absence (middle) or presence (right) of PG545 pretreatment. Pancreas tissues were collected 24 h thereafter, and 5 micron sections from formalin-fixed, paraffin-embedded samples were stained for LC-3 (marker of autophagy). Shown are representative photomicrographs at original magnification of x40. Note that LC-3 staining resembles the patchy pattern of heparanase staining (Fig. 1D,E ).

    Article Snippet: Slides were incubated with 10% normal goat serum (NGS) in phosphate buffered saline (PBS) for 60 min to block nonspecific binding and incubated overnight at 4 °C with antibodies directed against LC-3 (Cell signaling #2775), cathepsin L (1:200, R & D systems, #AF1515), heparanase (1:150, #733 raised against the N terminus of the 50 kDa heparanase subunit) , p65 NFκB subunit (1:100, Santa Cruz Biotechnology, sc-372), and pSTAT3 (1:100, Santa Cruz Biotechnology, sc-8059) diluted in blocking solution.

    Techniques: Staining, Mouse Assay, Injection, Formalin-fixed Paraffin-Embedded, Electron Microscopy, Immunostaining, Marker

    hTERT regulates the transcriptional activity of heparanase through the c-Myc binding site A. The 5′-flanking promoter region of the heparanase gene was cloned into the NheI/HindIII site of the luciferase construct pGL3-Basic vector. B. The relative luciferase activities of the heparanase promoter were tested using the dual luciferase assay in SGC7901 and MKN45 cells that were transiently transfected with pIRES2-hTERT (50, 200, 500 ng) and sh-hTERT-1 and sh-hTERT-2 vectors, respectively. C. The putative binding site of c-Myc on the heparanase promoter region was predicted using bioinformatics analysis. D. Chromatin immunoprecipitation (ChIP) assays were conducted in SGC7901 and MKN45 cells using an antibody against c-Myc or a control rabbit normal immunoglobulin G. An equivalent of amount of DNA in all of the samples served as an Input control. E. The luciferase activities of the heparanase promoter with wild type (or the mutant binding site of c-Myc) were determined using luciferase reporter assays in SGC7901 and MKN45 cells that were transiently transfected with pIRES2, pIRES2-hTERT plasmids. (* P

    Journal: Oncotarget

    Article Title: Human telomerase reverse transcriptase (hTERT) promotes gastric cancer invasion through cooperating with c-Myc to upregulate heparanase expression

    doi: 10.18632/oncotarget.6575

    Figure Lengend Snippet: hTERT regulates the transcriptional activity of heparanase through the c-Myc binding site A. The 5′-flanking promoter region of the heparanase gene was cloned into the NheI/HindIII site of the luciferase construct pGL3-Basic vector. B. The relative luciferase activities of the heparanase promoter were tested using the dual luciferase assay in SGC7901 and MKN45 cells that were transiently transfected with pIRES2-hTERT (50, 200, 500 ng) and sh-hTERT-1 and sh-hTERT-2 vectors, respectively. C. The putative binding site of c-Myc on the heparanase promoter region was predicted using bioinformatics analysis. D. Chromatin immunoprecipitation (ChIP) assays were conducted in SGC7901 and MKN45 cells using an antibody against c-Myc or a control rabbit normal immunoglobulin G. An equivalent of amount of DNA in all of the samples served as an Input control. E. The luciferase activities of the heparanase promoter with wild type (or the mutant binding site of c-Myc) were determined using luciferase reporter assays in SGC7901 and MKN45 cells that were transiently transfected with pIRES2, pIRES2-hTERT plasmids. (* P

    Article Snippet: The transferred membranes were subsequently incubated with primary rabbit anti-hTERT, anti-c-Myc, anti-heparanase, anti-Cyclin D1 (Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA) antibodies.

    Techniques: Activity Assay, Binding Assay, Clone Assay, Luciferase, Construct, Plasmid Preparation, Transfection, Chromatin Immunoprecipitation, Mutagenesis

    hTERT interacts with c-Myc and regulates the binding of the hTERT/c-Myc complex to the heparanase promoter A. The total cell lysates of SGC7901 cells were prepared for immunoprecipitation (IP) with antibodies against hTERT or c-Myc, respectively. Rabbit IgG served as a negative control. An aliquot of cell lysate was used as an Input control. B. Cytoplasm and nuclear lysates of SGC7901 cells were prepared for IP with anti-hTERT or anti-c-Myc and then evaluated by IB. C. The subcellular localization and the colocalization of hTERT and c-Myc were tested by dual immunofluorescence using confocal microscopy. D. hTERT and c-Myc co-existed in the heparanase promoter region. ChIP assays were performed using antibody against c-Myc or hTERT. E. Binding of the c-Myc/hTERT complex to promoter was mediated by c-Myc. SGC7901 cells were transfected with sh-c-Myc vectors, and then ChIP was performed using an antibody against c-Myc or hTERT. F. hTERT and c-Myc were present in the same protein complex on the heparanase promoter. Soluble chromatin from SGC7901 cells was first incubated with anti-c-Myc or anti-hTERT antibodies. The ReChIP assay was similarly processed using anti-hTERT or anti-c-Myc, respectively. G. hTERT regulates the interaction of c-Myc and hTERT. Co-IP was performed and evaluated by IB with the indicated antibodies. H. hTERT regulates the recruitment of c-Myc to the heparanase promoter. SGC7901 cells were transfected with vectors as indicated. Soluble chromatin was prepared for the ChIP assays.

    Journal: Oncotarget

    Article Title: Human telomerase reverse transcriptase (hTERT) promotes gastric cancer invasion through cooperating with c-Myc to upregulate heparanase expression

    doi: 10.18632/oncotarget.6575

    Figure Lengend Snippet: hTERT interacts with c-Myc and regulates the binding of the hTERT/c-Myc complex to the heparanase promoter A. The total cell lysates of SGC7901 cells were prepared for immunoprecipitation (IP) with antibodies against hTERT or c-Myc, respectively. Rabbit IgG served as a negative control. An aliquot of cell lysate was used as an Input control. B. Cytoplasm and nuclear lysates of SGC7901 cells were prepared for IP with anti-hTERT or anti-c-Myc and then evaluated by IB. C. The subcellular localization and the colocalization of hTERT and c-Myc were tested by dual immunofluorescence using confocal microscopy. D. hTERT and c-Myc co-existed in the heparanase promoter region. ChIP assays were performed using antibody against c-Myc or hTERT. E. Binding of the c-Myc/hTERT complex to promoter was mediated by c-Myc. SGC7901 cells were transfected with sh-c-Myc vectors, and then ChIP was performed using an antibody against c-Myc or hTERT. F. hTERT and c-Myc were present in the same protein complex on the heparanase promoter. Soluble chromatin from SGC7901 cells was first incubated with anti-c-Myc or anti-hTERT antibodies. The ReChIP assay was similarly processed using anti-hTERT or anti-c-Myc, respectively. G. hTERT regulates the interaction of c-Myc and hTERT. Co-IP was performed and evaluated by IB with the indicated antibodies. H. hTERT regulates the recruitment of c-Myc to the heparanase promoter. SGC7901 cells were transfected with vectors as indicated. Soluble chromatin was prepared for the ChIP assays.

    Article Snippet: The transferred membranes were subsequently incubated with primary rabbit anti-hTERT, anti-c-Myc, anti-heparanase, anti-Cyclin D1 (Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA) antibodies.

    Techniques: Binding Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Confocal Microscopy, Chromatin Immunoprecipitation, Transfection, Incubation, Co-Immunoprecipitation Assay

    The intact hTERT and c-Myc complex is essential for heparanase promoter activity and expression A. and B. hTERT knockdown impaired c-Myc-enhanced heparanase promoter activities and protein expression. SGC7901 and MKN45 cells were transiently transfected with pEX-3 or c-Myc plasmids, and sh-hTERT-1 or sh-hTERT-2 as indicated, respectively. The relative luciferase activities of the heparanase promoter and protein expression were examined using the dual luciferase assay and western blot analysis. (* P

    Journal: Oncotarget

    Article Title: Human telomerase reverse transcriptase (hTERT) promotes gastric cancer invasion through cooperating with c-Myc to upregulate heparanase expression

    doi: 10.18632/oncotarget.6575

    Figure Lengend Snippet: The intact hTERT and c-Myc complex is essential for heparanase promoter activity and expression A. and B. hTERT knockdown impaired c-Myc-enhanced heparanase promoter activities and protein expression. SGC7901 and MKN45 cells were transiently transfected with pEX-3 or c-Myc plasmids, and sh-hTERT-1 or sh-hTERT-2 as indicated, respectively. The relative luciferase activities of the heparanase promoter and protein expression were examined using the dual luciferase assay and western blot analysis. (* P

    Article Snippet: The transferred membranes were subsequently incubated with primary rabbit anti-hTERT, anti-c-Myc, anti-heparanase, anti-Cyclin D1 (Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA) antibodies.

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

    hTERT promotes the invasion and metastasis of gastric cancer cells through c-Myc and heparanase in vitro and in vivo A. c-Myc and heparanase knockdown impaired hTERT-enhanced invasion of gastric cancer cells in vitro . The invasive properties of the cells were analyzed using an invasion assay with a Matrigel-coated plate. All of the experiments were performed at least three times ( P

    Journal: Oncotarget

    Article Title: Human telomerase reverse transcriptase (hTERT) promotes gastric cancer invasion through cooperating with c-Myc to upregulate heparanase expression

    doi: 10.18632/oncotarget.6575

    Figure Lengend Snippet: hTERT promotes the invasion and metastasis of gastric cancer cells through c-Myc and heparanase in vitro and in vivo A. c-Myc and heparanase knockdown impaired hTERT-enhanced invasion of gastric cancer cells in vitro . The invasive properties of the cells were analyzed using an invasion assay with a Matrigel-coated plate. All of the experiments were performed at least three times ( P

    Article Snippet: The transferred membranes were subsequently incubated with primary rabbit anti-hTERT, anti-c-Myc, anti-heparanase, anti-Cyclin D1 (Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA) antibodies.

    Techniques: In Vitro, In Vivo, Invasion Assay

    hTERT correlates significantly with heparanase in GC tissues and regulates heparanase expression in GC cells A. Immunohistochemical staining for the expression of hTERT and heparanase in gastric cancer and corresponding adjacent normal tissues. B. and C. Semi-quantitative analysis used to score the tissue sections, and relative expression levels of hTERT and heparanase determined in gastric cancer and corresponding adjacent normal tissues. D. Correlation of the protein expression levels of hTERT and heparanase were analyzed using Pearson's correlation analysis ( P

    Journal: Oncotarget

    Article Title: Human telomerase reverse transcriptase (hTERT) promotes gastric cancer invasion through cooperating with c-Myc to upregulate heparanase expression

    doi: 10.18632/oncotarget.6575

    Figure Lengend Snippet: hTERT correlates significantly with heparanase in GC tissues and regulates heparanase expression in GC cells A. Immunohistochemical staining for the expression of hTERT and heparanase in gastric cancer and corresponding adjacent normal tissues. B. and C. Semi-quantitative analysis used to score the tissue sections, and relative expression levels of hTERT and heparanase determined in gastric cancer and corresponding adjacent normal tissues. D. Correlation of the protein expression levels of hTERT and heparanase were analyzed using Pearson's correlation analysis ( P

    Article Snippet: The transferred membranes were subsequently incubated with primary rabbit anti-hTERT, anti-c-Myc, anti-heparanase, anti-Cyclin D1 (Santa Cruz, CA, USA) and mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA) antibodies.

    Techniques: Expressing, Immunohistochemistry, Staining

    Structures docked into human heparanase: (A) diantennary monomer 6 , (B) monoantennary monomer 5 , (C) hydrogen bonding network of disaccharide of dianntennary monomer 6 in heparanase subsites −1/−2, (D) hydrogen boding monosaccharide of dianntennary monomer 6 to HBD-2, and (E) hydrogen bonding network of disaccharide of monoanntennary monomer 5 in heparanase subsites −1/−2. Hydrogen bonding shown in magenta. HBD-2 is on the left and HBD-1 is on the right.

    Journal: Biomacromolecules

    Article Title: Glycosidase Inhibition by Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymers

    doi: 10.1021/acs.biomac.7b01049

    Figure Lengend Snippet: Structures docked into human heparanase: (A) diantennary monomer 6 , (B) monoantennary monomer 5 , (C) hydrogen bonding network of disaccharide of dianntennary monomer 6 in heparanase subsites −1/−2, (D) hydrogen boding monosaccharide of dianntennary monomer 6 to HBD-2, and (E) hydrogen bonding network of disaccharide of monoanntennary monomer 5 in heparanase subsites −1/−2. Hydrogen bonding shown in magenta. HBD-2 is on the left and HBD-1 is on the right.

    Article Snippet: A total of 42 μ L of inhibitor solution in Milli-Q water (0.00016–4000 μ M) or just Milli-Q water (as a control) and 42 μ L of heparanase (5.3 nM, R & D Systems) solution in pH 7.5 tris buffer (consisting of 20 mM Tris HCl, 0.15 M NaCl, and 0.1% CHAPS) or just buffer as a blank were added into microtubes and preincubated at 37 °C for 10 min. Next, 84 μ L of biotin-heparan sulfate-Eu cryptate (Cisbio, Cat # 61BHSKAA; 58.6 ng in pH 5.5 0.2 M NaCH3 CO2 buffer) was added to the microtubes, and the resulting mixture was incubated for 60 min at 37 °C.

    Techniques:

    Hydrolytic potential over time of heparanase on monoantennary and diantennary polymers and Fondaparinux.

    Journal: Biomacromolecules

    Article Title: Glycosidase Inhibition by Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymers

    doi: 10.1021/acs.biomac.7b01049

    Figure Lengend Snippet: Hydrolytic potential over time of heparanase on monoantennary and diantennary polymers and Fondaparinux.

    Article Snippet: A total of 42 μ L of inhibitor solution in Milli-Q water (0.00016–4000 μ M) or just Milli-Q water (as a control) and 42 μ L of heparanase (5.3 nM, R & D Systems) solution in pH 7.5 tris buffer (consisting of 20 mM Tris HCl, 0.15 M NaCl, and 0.1% CHAPS) or just buffer as a blank were added into microtubes and preincubated at 37 °C for 10 min. Next, 84 μ L of biotin-heparan sulfate-Eu cryptate (Cisbio, Cat # 61BHSKAA; 58.6 ng in pH 5.5 0.2 M NaCH3 CO2 buffer) was added to the microtubes, and the resulting mixture was incubated for 60 min at 37 °C.

    Techniques:

    Schematic of cell migration and angiogenesis by MSC-secreted heparanase. Abbreviation: MSC, mesenchymal stem cell.

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

    doi: 10.1002/stem.1995

    Figure Lengend Snippet: Schematic of cell migration and angiogenesis by MSC-secreted heparanase. Abbreviation: MSC, mesenchymal stem cell.

    Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R & D Systems, Minneapolis, MN, USA).

    Techniques: Migration

    Proangiogenic role of MSC hpa in vitro. (A) : Protein expression of heparanase and actin by Western blot in MSC null and MSC hpa-KD . (B) : Detection of heparanase concentration using ELISA in conditioned medium of MSC WT , MSC null , and MSC hpa-KD ; n = 4 for each group, * denotes p

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

    doi: 10.1002/stem.1995

    Figure Lengend Snippet: Proangiogenic role of MSC hpa in vitro. (A) : Protein expression of heparanase and actin by Western blot in MSC null and MSC hpa-KD . (B) : Detection of heparanase concentration using ELISA in conditioned medium of MSC WT , MSC null , and MSC hpa-KD ; n = 4 for each group, * denotes p

    Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R & D Systems, Minneapolis, MN, USA).

    Techniques: In Vitro, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    HIF-2 α activation by heparanase requires integrin β 1. (A) : Western blot and quantification of integrin β 1 and β 3 expressions in HUVECs treated with conditioned medium from MSC null , MSC hpa , and MSC hpa-KD . (B) : HIF-2 α and integrin β 1 expressions are decreased in integrin β 1 knockdown HUVECs. (C) : Representative images and bar graph of cell migration in HUVECs transfected with vector or integrin β 1 shRNA lentivirus; Bar = 50 μm, n = 3, ** denotes p

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

    doi: 10.1002/stem.1995

    Figure Lengend Snippet: HIF-2 α activation by heparanase requires integrin β 1. (A) : Western blot and quantification of integrin β 1 and β 3 expressions in HUVECs treated with conditioned medium from MSC null , MSC hpa , and MSC hpa-KD . (B) : HIF-2 α and integrin β 1 expressions are decreased in integrin β 1 knockdown HUVECs. (C) : Representative images and bar graph of cell migration in HUVECs transfected with vector or integrin β 1 shRNA lentivirus; Bar = 50 μm, n = 3, ** denotes p

    Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R & D Systems, Minneapolis, MN, USA).

    Techniques: Activation Assay, Western Blot, Migration, Transfection, Plasmid Preparation, shRNA

    Comparison of splice 5 (S5) and wild-type (WT) human heparanase amino acid sequences (GCG alignment program). The two catalytic Glu residues, the proton donor and nucleophile, are marked in bold. Note that the proton donor is missing in splice 5. Potential

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Comparison of splice 5 (S5) and wild-type (WT) human heparanase amino acid sequences (GCG alignment program). The two catalytic Glu residues, the proton donor and nucleophile, are marked in bold. Note that the proton donor is missing in splice 5. Potential

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques:

    Nucleotide and predicted amino acid sequences of splice 5 human heparanase. Nucleotide sequences are shown above the predicted amino acid sequences. Numbers on the left correspond to nucleotides (Roman) and amino acid residues (bold italic). The signal

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Nucleotide and predicted amino acid sequences of splice 5 human heparanase. Nucleotide sequences are shown above the predicted amino acid sequences. Numbers on the left correspond to nucleotides (Roman) and amino acid residues (bold italic). The signal

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques:

    Cloning and expression of human heparanase splice variant lacking exon 5. A. Semi-quantitive RT-PCR using primers located around the human heparanase cDNA region encoded by exon 5. Bands of 579 bp represent the wild type enzyme, while those of 405 bp

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Cloning and expression of human heparanase splice variant lacking exon 5. A. Semi-quantitive RT-PCR using primers located around the human heparanase cDNA region encoded by exon 5. Bands of 579 bp represent the wild type enzyme, while those of 405 bp

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques: Clone Assay, Expressing, Variant Assay, Reverse Transcription Polymerase Chain Reaction

    Localization of Human Heparanase and its splice variant in glioma cells. U87 cells stably transfected with the full length wild type (WT) human heparanase or its splice variant #5 (S5), were immunostained with monoclonal anti-heparanase antibody. Note

    Journal:

    Article Title: Cloning, expression and characterization of an alternatively spliced variant of human heparanase

    doi: 10.1016/j.bbrc.2006.12.189

    Figure Lengend Snippet: Localization of Human Heparanase and its splice variant in glioma cells. U87 cells stably transfected with the full length wild type (WT) human heparanase or its splice variant #5 (S5), were immunostained with monoclonal anti-heparanase antibody. Note

    Article Snippet: Briefly, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with anti-heparanase monoclonal antibody [ ] (kindly provided by Dr. Hua-Quan Miao, Imclone Systems Inc, New York, N.Y).

    Techniques: Variant Assay, Stable Transfection, Transfection