Journal: The Journal of Biological Chemistry

Article Title: Heparanase and Syndecan-1 Interplay Orchestrates Fibroblast Growth Factor-2-induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

doi: 10.1074/jbc.M111.279836

Figure Lengend Snippet: Morphological changes and migration induced by FGF-2. A, representative images of WT and HPSE-silenced HK2 cells grown on a glass surface in serum-free condition for 5 days with or without FGF-2 (10 ng/ml). First lane, images of morphological changes at optical microscopy (magnification, ×200). Second lane, F-actin cell organization visualized by Alexa Fluor 350-conjugated phalloidin (magnification, ×600). B, left, representative images of scratch assays on confluent monolayers of WT and silenced HK2 cells obtained using a micropipette tip and adding FGF-2 (10 ng/ml) as indicated. The areas of cell-free wounds were photographed after 24 h of treatment. Right, histogram representing migration (in micrometers) over 24 h. Data are presented as mean ± S.D. (error bars) of three separate experiments. **, p < 0.001 versus WT control cells.

Article Snippet: A stably HPSE-silenced HK2 cell line was obtained by transfection with shRNA plasmid targeting human HPSE ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006665","term_id":"315360641","term_text":"NM_006665"}} NM_006665 ) purchased from OriGene, as recently described elsewhere.

Techniques: Migration, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Heparanase and Syndecan-1 Interplay Orchestrates Fibroblast Growth Factor-2-induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

doi: 10.1074/jbc.M111.279836

Figure Lengend Snippet: HPSE expression and activity regulation by FGF-2. A, HPSE and mRNA expression levels in WT and silenced HK2 cells determined by real-time PCR. WT and silenced cell lines were cultured with FGF-2 (10 ng/ml) for 6 h. The results were normalized using GAPDH as an internal control and represent the mean ± S.D. (error bars) of three separate experiments performed in triplicate. **, p < 0.001 and *, p < 0.05 versus WT control cells. B and C, HPSE activity in protein extracts (B) and serum-free conditioned medium (C) in WT and HPSE-silenced HK2 cells. The cells were treated with 10 ng/ml FGF-2 (open bars) in serum-free medium (filled bars). HPSE activity is expressed as nanograms of HS removed per minute. The results represent the mean ± S.D. of two separate experiments performed in triplicate. Platelet extract was used as a positive control (data not shown).**, p < 0.001 versus HK2 WT control.

Article Snippet: A stably HPSE-silenced HK2 cell line was obtained by transfection with shRNA plasmid targeting human HPSE ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006665","term_id":"315360641","term_text":"NM_006665"}} NM_006665 ) purchased from OriGene, as recently described elsewhere.

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Cell Culture, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Heparanase and Syndecan-1 Interplay Orchestrates Fibroblast Growth Factor-2-induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

doi: 10.1074/jbc.M111.279836

Figure Lengend Snippet: Role of HPSE activity. α-SMA, MMP-9, VIM, FN, HPSE, and SDC1 mRNA expression levels in WT HK2 cells cultured with and without FGF-2 and heparin were determined by real-time PCR. The results were normalized using GAPDH as an internal control and represent the mean ± S.D. (error bars) of two separate experiments performed in triplicate. **, p < 0.001 versus WT cells treated with FGF-2.

Article Snippet: A stably HPSE-silenced HK2 cell line was obtained by transfection with shRNA plasmid targeting human HPSE ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006665","term_id":"315360641","term_text":"NM_006665"}} NM_006665 ) purchased from OriGene, as recently described elsewhere.

Techniques: Activity Assay, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Heparanase and Syndecan-1 Interplay Orchestrates Fibroblast Growth Factor-2-induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

doi: 10.1074/jbc.M111.279836

Figure Lengend Snippet: FGF-2 regulates HPSE and SDC1 markers via the PI3K/AKT pathway. HPSE (A) and SDC1 (B) gene expression was quantified by real-time PCR. The analysis was performed in WT HK2 cells, preincubated with or without wortmannin (1 μm), and in AKT-silenced HK2 cells. The results were normalized using GAPDH as an internal control and represent the mean ± S.D. (error bars) of three separate experiments performed in triplicate. *, p < 0.05 versus WT control cells; ##, p < 0.001 and #, p < 0.05 versus WT + FGF-2 control cells.

Article Snippet: A stably HPSE-silenced HK2 cell line was obtained by transfection with shRNA plasmid targeting human HPSE ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006665","term_id":"315360641","term_text":"NM_006665"}} NM_006665 ) purchased from OriGene, as recently described elsewhere.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Heparanase and Syndecan-1 Interplay Orchestrates Fibroblast Growth Factor-2-induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

doi: 10.1074/jbc.M111.279836

Figure Lengend Snippet: HAT involvement. A, HAT activity in nuclear extract isolated from WT and HPSE-silenced cells with and without FGF-2 treatment. B, α-SMA, MMP-9, VIM, FN, HPSE, and SDC1 mRNA expression levels in WT HK2 cells cultured with and without FGF-2 and anacardic acid (HAT inhibitor) determined by real-time PCR. The results were normalized using GAPDH as an internal control and represent the mean ± S.D. (error bars) of two separate experiments performed in triplicate. ##, p < 0.001 versus WT control cells; **, p < 0.001 versus WT cells treated with FGF-2.

Article Snippet: A stably HPSE-silenced HK2 cell line was obtained by transfection with shRNA plasmid targeting human HPSE ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006665","term_id":"315360641","term_text":"NM_006665"}} NM_006665 ) purchased from OriGene, as recently described elsewhere.

Techniques: Activity Assay, Isolation, Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: A New Synthesized Dicarboxylated Oxy-Heparin Efficiently Attenuates Tumor Growth and Metastasis

doi: 10.3390/cells13030211

Figure Lengend Snippet: Heparanase-inhibiting activity of compound XII. ( A ) Recombinant heparanase (150 ng/mL) was incubated (18 h, 37 °C) with fondaparinux (Arixtra) in the absence and presence of increasing concentrations (12.5–800 ng/mL) of compound XII. Cleavage of the pentasaccharide substrate into disaccharide and trisaccharide products was colorimetrically evaluated at 584 nm following the addition of WST-1. ( B ) Increasing concentrations of compound XII were incubated (4 h) with recombinant heparanase and biotin-labeled HS substrate (CisBio). Heparanase enzymatic activity was determined by the FRET assay, as described in , yielding an IC50 = 241 ng/mL. ( C ) Inhibition of ECM degradation by XII. Recombinant heparanase (2 ng/mL) was incubated (4 h, 37 °C) with sulfate-labeled ECM in the absence and presence of 0.2 and 1.0 µg/mL of compound XII or SST0001 (= Roneparstat). Sulfate-labeled HS degradation products released into the incubation medium were subjected to gel filtration on Sepharose 6B.

Article Snippet: Western blot analysis was carried out using anti-heparanase antibody (Proteintech 66226-1-ig, Rosemont, IL, USA) and anti-GAPDH polyclonal antibodies.

Techniques: Activity Assay, Recombinant, Incubation, Labeling, Inhibition, Filtration

Journal: Cells

Article Title: A New Synthesized Dicarboxylated Oxy-Heparin Efficiently Attenuates Tumor Growth and Metastasis

doi: 10.3390/cells13030211

Figure Lengend Snippet: Heparanase protein expression, uptake, and processing. ( A ) Compound XII attenuates heparanase levels in PDAC tumors. Nude mice (n = 6/group) s.c. inoculated with Mia PaCa-2 cells were treated with increasing amounts of compound XII (daily injections of 20, 40, or 80 mg/kg) or vehicle alone, as described in . On Day 25, the tumors were resected and the tumor tissue lysates were subjected to Western blotting, applying anti-heparanase (Proteintech 66226-1-ig, Rosemont, IL, USA) and anti-GAPDH polyclonal antibodies. The positive control (HPA 220408 = GS3-linker 58 kDa) was the purified recombinant protein used in the FRET activity assay. The detected endogenous heparanase was presumably the 50 kDa subunit. The molecular weight of standard protein (ladder) was indicated. Treatment with compound XII (20 mg/kg/day) resulted in a marked 50% decrease in the normalized (GAPDH) amount of heparanase. There were no statistical differences between the different doses of compound XII. ( B ) Compound XII inhibits cellular uptake and processing of exogenously added heparanase. Cervical carcinoma cells (SiHa) were left untreated (Con) or were incubated with latent 65 kDa heparanase (1 ug/mL) together with increasing concentrations (1, 5, 10 ug/mL) of compound XII. After 24 h, cultures were washed, and cell lysate samples were subjected to immunoblotting, applying anti-heparanase (upper panel = 50 kDa heparanase subunit) and anti-actin (lower panel) antibodies. The polyclonal anti-heparanase antibodies (ANT-155) were from ProSpec-Tany, TechnoGene Ltd., Ness-Ziona, Israel).

Article Snippet: Western blot analysis was carried out using anti-heparanase antibody (Proteintech 66226-1-ig, Rosemont, IL, USA) and anti-GAPDH polyclonal antibodies.

Techniques: Expressing, Western Blot, Positive Control, Purification, Recombinant, Activity Assay, Molecular Weight, Incubation

Journal: Carbohydrate polymers

Article Title: Polymeric Fluorescent Heparin as One-Step FRET Substrate of Human Heparanase

doi: 10.1016/j.carbpol.2018.10.071

Figure Lengend Snippet: Generic structure of heparin (R = H or SO3−; R1 = H, COCH3 or SO3−) showing typical site (↓) cleaved by human heparanase (HPSE).

Article Snippet: Antibodies used for analysis include anti-heparanase 1 (aa 301-331, Boster bio, PB9427) and anti-HPSE (aa34-115, LS Bio, LS-C294467).

Techniques:

Journal: Carbohydrate polymers

Article Title: Polymeric Fluorescent Heparin as One-Step FRET Substrate of Human Heparanase

doi: 10.1016/j.carbpol.2018.10.071

Figure Lengend Snippet: FRET-based assay for human heparanase. Heparin-DE (1 mg/ml) was incubated with HPSE (1 μM) at 37°C in 20 mM sodium acetate buffer, pH 5.0. Fluorescence emission spectra (λEX = 340 nm) at (bold line) 0 h, (dotted line) 4 h.

Article Snippet: Antibodies used for analysis include anti-heparanase 1 (aa 301-331, Boster bio, PB9427) and anti-HPSE (aa34-115, LS Bio, LS-C294467).

Techniques: Incubation, Fluorescence

Journal: Carbohydrate polymers

Article Title: Polymeric Fluorescent Heparin as One-Step FRET Substrate of Human Heparanase

doi: 10.1016/j.carbpol.2018.10.071

Figure Lengend Snippet: Optimization of heparanase assay using heparin-DE as substrate. (A) Labeled heparin (1 mg/ml) was incubated 1 μM HPSE at 37 °C for indicated times followed by measurement of emission at 500 nm. (B) Labeled heparin (1 mg/ml) was incubated with varying concentrations HPSE at 37 °C for 4 h followed by fluorescence measurement. (C) Michaelis–Menten kinetics of HPSE cleavage of heparin–DE using the optimized FRET quenching assay. The cleavage reactions were performed in microplate format (100 μL) in 20 mM sodium acetate buffer, pH 5.0, containing 1 mg/mL heparin–DE and 1 μM HPSE at 37 °C for 4 h. Solid lines represent curve fitting to the standard Michaelis equation. Error bars show variation from at least 3 measurements. F and F0 are fluorescence signals corresponding to the test sample and blank, respectively.

Article Snippet: Antibodies used for analysis include anti-heparanase 1 (aa 301-331, Boster bio, PB9427) and anti-HPSE (aa34-115, LS Bio, LS-C294467).

Techniques: Labeling, Incubation, Fluorescence

Journal: Carbohydrate polymers

Article Title: Polymeric Fluorescent Heparin as One-Step FRET Substrate of Human Heparanase

doi: 10.1016/j.carbpol.2018.10.071

Figure Lengend Snippet: Suramin inhibition of HPSE using the one-step FRET quenching assay. The experiments were performed in microplate format (100 μL) in 20 mM sodium acetate buffer, pH 5.0, containing 1 mg/mL heparin–DE and 1 μM HPSE at 37 °C for 4 h in the presence of varying concentrations of suramin. Solid lines represent curve fitting to standard dose-response equation.

Article Snippet: Antibodies used for analysis include anti-heparanase 1 (aa 301-331, Boster bio, PB9427) and anti-HPSE (aa34-115, LS Bio, LS-C294467).

Techniques: Inhibition

Journal: Carbohydrate polymers

Article Title: Polymeric Fluorescent Heparin as One-Step FRET Substrate of Human Heparanase

doi: 10.1016/j.carbpol.2018.10.071

Figure Lengend Snippet: Expression of active heparanase by MCF7 cells under normoxic conditions. Inset shows the HEK cells expressed HPSE activity measurement. Results are presented as the mean±SD (n>3).

Article Snippet: Antibodies used for analysis include anti-heparanase 1 (aa 301-331, Boster bio, PB9427) and anti-HPSE (aa34-115, LS Bio, LS-C294467).

Techniques: Expressing, Activity Assay

Journal: BioMed Research International

Article Title: Molecular Mechanism of Gleditsiae Spina for the Treatment of High-Grade Serous Ovarian Cancer Based on Network Pharmacology and Pharmacological Experiments

doi: 10.1155/2022/5988310

Figure Lengend Snippet: The cellular verification results. Gleditsiae Spina inhibited ovarian cancer cells proliferation in a dose-dependent way after incubation 24 h (a) and 48 h (b). (c) After exposure to the Gleditsiae Spina for 24 h, the total protein from each cell lysate was analyzed by Western blotting to measure the expression of proteins. (d) Data are presented as the mean ± SD and normalized to β -actin. (e) The AKT expression was decreased by AKT kinase inhibitor. (f) Dose-dependent inhibitory effect of Gleditsiae Spina on ovarian cancer cell proliferation under AKT kinase inhibitor of 2 nM ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001).

Article Snippet: The membranes were blocked with 5% BSA (Amresco, Solon, OH, USA) at room temperature for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-HPSE1 (1 : 1000, CST,USA), anti-MMP9 (1 : 1000, CST,USA), anti- β -catenin (1 : 1000, CST, USA), anti-N-cad (1 : 1000 CST, USA), anti-E-cad (1 : 1000 CST, USA),anti-PI3K/p-PI3K (1 : 1000, CST,USA), anti-AKT/p-AKT (1 : 1000, CST,USA), anti-YAP/TAZ (1 : 1000, CST,USA) and β -actin (1 : 10000, CST,USA).

Techniques: Incubation, Western Blot, Expressing