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  • 97
    Millipore vpc23019
    The analysis of the effect of <t>VPC23019</t> in the experimental asthma mouse model. OVA-sensitized mice were administered PBS or anti-CCL20 antibody (20 μg/mouse) via i.p. injection before OVA inhalation on days 21 and 22. At day 23, BALF was collected, and inflammatory cells were analyzed. Data are expressed as the mean ± SEM obtained from three mice per group (A). Lungs and BALF were collected one day after the last challenge of the OVA-sensitized mice with the aerosol containing OVA or with PBS alone. Some mice received an intraperitoneal injection of 1 μg VPC23019 in a volume of 500 μl sterile PBS 30 minutes before each OVA exposure. Lung sections were subjected to H E staining or PAS staining. Bars , 100 μm. PAS + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of PAS + epithelial cells was determined as 100 × (PAS + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (B). The total inflammatory cells in the BALF were counted. For differential inflammatory cells counting, inflammatory cells were spun down and stained with Diff-Quick. Data are expressed as the mean ± SEM obtained for three mice in each group (C). Paraffin-embedded sections of the lung were stained with the antibody against CCL20 ( brown ) CCL20 + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of CCL20 + epithelial cells was determined as 100 × (CCL20 + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (D). BEAS-2B cells were treated with S1P (1 μM) and VPC23019 (10 μM) (B) for 3 h, and CCL3, TIMP2, and IL-8 gene expressions were analyzed by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3) (E). *, P
    Vpc23019, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 12 article reviews
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    92
    Avanti Polar vpc23019
    Jurkat cell migration induced by S1P. (A) Jurkat cells were seeded into the upper well of the Transwell cell culture chambers, and allowed to migrate to the lower chamber, where various concentrations of S1P were placed, for 4 h. A bell-shape pattern of migration was observed, with a dose-dependent increase of migration with S1P from 0.1 – 100 nM, but decreasing with S1P 1 μM. (B) Jurkat cells, pretreated without (open column) or with (solid column) 200 ng/mL of PTX for 60 min, were allowed to migrate toward the indicated concentrations of 100 nM S1P (n = 4). PTX pretreatment inhibited the migration of Jurkat cells induced by S1P. Statistically significant compared to control cells, i.e., without PTX treatment. (C) Jurkat cells pretreated without (open column) or with 20 μM (solid column) or with 50 μM (hatched column) of <t>VPC23019</t> for 60 min were allowed to migrate toward 100 nM S1P (n = 3). VPC23019 at 50 μM, but not 20 μM, significantly inhibited migration of Jurkat cells induced by S1P. *Statistically significant compared to control cells, i.e., without VPC23019 treatment ( P
    Vpc23019, supplied by Avanti Polar, used in various techniques. Bioz Stars score: 92/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    94
    Tocris vpc23019
    Sphingosine-1-phosphate (S1P)-primed neutrophils for antineutrophil cytoplasmic antibody (ANCA)-induced respiratory burst and degranulation. (A) Neutrophil respiratory burst induced by patient-derived ANCA-positive IgG was measured by conversion of dihydrorhodamine-123 (DHR-123) to rhodamine-123 in S1P-primed cells in the presence and absence of CYM50358, <t>VPC23019</t> and FTY720, respectively. (B) ANCA-induced neutrophil degranulation was determined by measuring the lactoferrin concentrations in the supernatant of neutrophil degranulation reaction. CYM50358, VPC23019 and FTY720 reduced ANCA-induced lactoferrin release.
    Vpc23019, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
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    93
    Millipore reagents vpc23019
    The analysis of the effect of <t>VPC23019</t> in the experimental asthma mouse model. OVA-sensitized mice were administered PBS or anti-CCL20 antibody (20 μg/mouse) via i.p. injection before OVA inhalation on days 21 and 22. At day 23, BALF was collected, and inflammatory cells were analyzed. Data are expressed as the mean ± SEM obtained from three mice per group (A). Lungs and BALF were collected one day after the last challenge of the OVA-sensitized mice with the aerosol containing OVA or with PBS alone. Some mice received an intraperitoneal injection of 1 μg VPC23019 in a volume of 500 μl sterile PBS 30 minutes before each OVA exposure. Lung sections were subjected to H E staining or PAS staining. Bars , 100 μm. PAS + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of PAS + epithelial cells was determined as 100 × (PAS + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (B). The total inflammatory cells in the BALF were counted. For differential inflammatory cells counting, inflammatory cells were spun down and stained with Diff-Quick. Data are expressed as the mean ± SEM obtained for three mice in each group (C). Paraffin-embedded sections of the lung were stained with the antibody against CCL20 ( brown ) CCL20 + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of CCL20 + epithelial cells was determined as 100 × (CCL20 + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (D). BEAS-2B cells were treated with S1P (1 μM) and VPC23019 (10 μM) (B) for 3 h, and CCL3, TIMP2, and IL-8 gene expressions were analyzed by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3) (E). *, P
    Reagents Vpc23019, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology vpc 23019
    Proposed scheme for S1P regulation of adipocyte proliferation and differentiation by the S1pr1/S1pr3–S1pr2 axis. JTE-013 was used as a S1pr2-specific antagonist and <t>VPC-23019</t> as a S1pr1/S1pr3-specific antagonist.
    Vpc 23019, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
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    90
    Cayman Chemical vpc23019
    COX-2 expression and PGI-2 release induced by HDL were inhibited by different S1P receptors (S1PRs) inhibitors in HUVECs. a – c Cells were washed and pretreated with PBS or the S1P1 and S1P3 inhibitor <t>VPC23019</t> (10 μmol/L), or the S1P2 inhibitor JTE013 (10 μmol/L) for 1 h, and then stimulated without or with 120 μg/ml HDL for 8 h. The expression of COX-2 was assayed by Western blot analysis ( a ), relative protein expression of COX-2 was normalized to GAPDH ( b ),and the production of PGI-2 was determined by competitive ELISA ( c ). Data were mean ± SE from three separate experiments. * P
    Vpc23019, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    The analysis of the effect of VPC23019 in the experimental asthma mouse model. OVA-sensitized mice were administered PBS or anti-CCL20 antibody (20 μg/mouse) via i.p. injection before OVA inhalation on days 21 and 22. At day 23, BALF was collected, and inflammatory cells were analyzed. Data are expressed as the mean ± SEM obtained from three mice per group (A). Lungs and BALF were collected one day after the last challenge of the OVA-sensitized mice with the aerosol containing OVA or with PBS alone. Some mice received an intraperitoneal injection of 1 μg VPC23019 in a volume of 500 μl sterile PBS 30 minutes before each OVA exposure. Lung sections were subjected to H E staining or PAS staining. Bars , 100 μm. PAS + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of PAS + epithelial cells was determined as 100 × (PAS + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (B). The total inflammatory cells in the BALF were counted. For differential inflammatory cells counting, inflammatory cells were spun down and stained with Diff-Quick. Data are expressed as the mean ± SEM obtained for three mice in each group (C). Paraffin-embedded sections of the lung were stained with the antibody against CCL20 ( brown ) CCL20 + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of CCL20 + epithelial cells was determined as 100 × (CCL20 + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (D). BEAS-2B cells were treated with S1P (1 μM) and VPC23019 (10 μM) (B) for 3 h, and CCL3, TIMP2, and IL-8 gene expressions were analyzed by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3) (E). *, P

    Journal: PLoS ONE

    Article Title: Role of S1P/S1PR3 axis in release of CCL20 from human bronchial epithelial cells

    doi: 10.1371/journal.pone.0203211

    Figure Lengend Snippet: The analysis of the effect of VPC23019 in the experimental asthma mouse model. OVA-sensitized mice were administered PBS or anti-CCL20 antibody (20 μg/mouse) via i.p. injection before OVA inhalation on days 21 and 22. At day 23, BALF was collected, and inflammatory cells were analyzed. Data are expressed as the mean ± SEM obtained from three mice per group (A). Lungs and BALF were collected one day after the last challenge of the OVA-sensitized mice with the aerosol containing OVA or with PBS alone. Some mice received an intraperitoneal injection of 1 μg VPC23019 in a volume of 500 μl sterile PBS 30 minutes before each OVA exposure. Lung sections were subjected to H E staining or PAS staining. Bars , 100 μm. PAS + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of PAS + epithelial cells was determined as 100 × (PAS + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (B). The total inflammatory cells in the BALF were counted. For differential inflammatory cells counting, inflammatory cells were spun down and stained with Diff-Quick. Data are expressed as the mean ± SEM obtained for three mice in each group (C). Paraffin-embedded sections of the lung were stained with the antibody against CCL20 ( brown ) CCL20 + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of CCL20 + epithelial cells was determined as 100 × (CCL20 + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (D). BEAS-2B cells were treated with S1P (1 μM) and VPC23019 (10 μM) (B) for 3 h, and CCL3, TIMP2, and IL-8 gene expressions were analyzed by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3) (E). *, P

    Article Snippet: VPC23019 (also known as BMS-345541; 401480, Calbiochem, Darmstadt, Germany) and JTE23019 (Cayman, Ann Arbor, MI) were commercially obtained.

    Techniques: Mouse Assay, Injection, Staining, Diff-Quik, Quantitative RT-PCR

    Jurkat cell migration induced by S1P. (A) Jurkat cells were seeded into the upper well of the Transwell cell culture chambers, and allowed to migrate to the lower chamber, where various concentrations of S1P were placed, for 4 h. A bell-shape pattern of migration was observed, with a dose-dependent increase of migration with S1P from 0.1 – 100 nM, but decreasing with S1P 1 μM. (B) Jurkat cells, pretreated without (open column) or with (solid column) 200 ng/mL of PTX for 60 min, were allowed to migrate toward the indicated concentrations of 100 nM S1P (n = 4). PTX pretreatment inhibited the migration of Jurkat cells induced by S1P. Statistically significant compared to control cells, i.e., without PTX treatment. (C) Jurkat cells pretreated without (open column) or with 20 μM (solid column) or with 50 μM (hatched column) of VPC23019 for 60 min were allowed to migrate toward 100 nM S1P (n = 3). VPC23019 at 50 μM, but not 20 μM, significantly inhibited migration of Jurkat cells induced by S1P. *Statistically significant compared to control cells, i.e., without VPC23019 treatment ( P

    Journal: Lipids in Health and Disease

    Article Title: Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells

    doi: 10.1186/1476-511X-13-150

    Figure Lengend Snippet: Jurkat cell migration induced by S1P. (A) Jurkat cells were seeded into the upper well of the Transwell cell culture chambers, and allowed to migrate to the lower chamber, where various concentrations of S1P were placed, for 4 h. A bell-shape pattern of migration was observed, with a dose-dependent increase of migration with S1P from 0.1 – 100 nM, but decreasing with S1P 1 μM. (B) Jurkat cells, pretreated without (open column) or with (solid column) 200 ng/mL of PTX for 60 min, were allowed to migrate toward the indicated concentrations of 100 nM S1P (n = 4). PTX pretreatment inhibited the migration of Jurkat cells induced by S1P. Statistically significant compared to control cells, i.e., without PTX treatment. (C) Jurkat cells pretreated without (open column) or with 20 μM (solid column) or with 50 μM (hatched column) of VPC23019 for 60 min were allowed to migrate toward 100 nM S1P (n = 3). VPC23019 at 50 μM, but not 20 μM, significantly inhibited migration of Jurkat cells induced by S1P. *Statistically significant compared to control cells, i.e., without VPC23019 treatment ( P

    Article Snippet: C17 -sphingosine 1-phosphate (C17 -S1P), lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and VPC23019 were purchased from Avanti Polar Lipids Inc (Alabaster, AL).

    Techniques: Migration, Cell Culture

    Induction of Jurkat cell migration by human serum and its reversal by VPC23019. Jurkat cells pretreated without (open column) or with (solid column) 50 μM of VPC23019 for 60 min were allowed to migrate toward the indicated concentrations of human serum (100-fold dilutions and 10-fold dilutions) (n = 3). Human serum induced strong and dose-dependent migration of Jurkat cells, and the migration induced by human serum at a 100-fold dilution was comparable to that induced by S1P at 100 nM. VPC23019 significantly inhibited the migratory response induced by S1P and human serum at 100-fold dilution, but not that induced by human serum at 10-fold dilution. *Statistically significant compared to cells without VPC23019 pretreatment ( P

    Journal: Lipids in Health and Disease

    Article Title: Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells

    doi: 10.1186/1476-511X-13-150

    Figure Lengend Snippet: Induction of Jurkat cell migration by human serum and its reversal by VPC23019. Jurkat cells pretreated without (open column) or with (solid column) 50 μM of VPC23019 for 60 min were allowed to migrate toward the indicated concentrations of human serum (100-fold dilutions and 10-fold dilutions) (n = 3). Human serum induced strong and dose-dependent migration of Jurkat cells, and the migration induced by human serum at a 100-fold dilution was comparable to that induced by S1P at 100 nM. VPC23019 significantly inhibited the migratory response induced by S1P and human serum at 100-fold dilution, but not that induced by human serum at 10-fold dilution. *Statistically significant compared to cells without VPC23019 pretreatment ( P

    Article Snippet: C17 -sphingosine 1-phosphate (C17 -S1P), lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and VPC23019 were purchased from Avanti Polar Lipids Inc (Alabaster, AL).

    Techniques: Migration

    Antagonist of S1P 1 and S1P 3 , but not S1P 2 , blocks HPAEC migration induced by S1P and S1PL silencing in a wound healing ECIS assay. HPAECs (∼50% confluence) grown on gold electrodes were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. Control and transfected cells were wounded on the gold electrodes as described under “Experimental Procedures” prior to VPC23019 (10 µM for 15 min) and following S1P (1.0 µM) challenge. Transendothelial electrical resistance (TER) was recorded for 16 h. Values are the mean ± S.E.M. for three independent experiments each performed in triplicates.

    Journal: PLoS ONE

    Article Title: Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

    doi: 10.1371/journal.pone.0016571

    Figure Lengend Snippet: Antagonist of S1P 1 and S1P 3 , but not S1P 2 , blocks HPAEC migration induced by S1P and S1PL silencing in a wound healing ECIS assay. HPAECs (∼50% confluence) grown on gold electrodes were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. Control and transfected cells were wounded on the gold electrodes as described under “Experimental Procedures” prior to VPC23019 (10 µM for 15 min) and following S1P (1.0 µM) challenge. Transendothelial electrical resistance (TER) was recorded for 16 h. Values are the mean ± S.E.M. for three independent experiments each performed in triplicates.

    Article Snippet: Inhibitors— VPC23019 were obtained from Avanti Polar Lipids (Alabaster, AL, USA).

    Techniques: Migration, Electric Cell-substrate Impedance Sensing, Transfection

    S1P-evoked ML-1 cell migration is inhibited by the S1P 1/3 antagonist VPC 23019 and the PI3K-inhibitor wortmannin

    Journal:

    Article Title: Sphingosine 1-phosphate receptor expression profile and regulation of migration in human thyroid cancer cells

    doi: 10.1042/BJ20060299

    Figure Lengend Snippet: S1P-evoked ML-1 cell migration is inhibited by the S1P 1/3 antagonist VPC 23019 and the PI3K-inhibitor wortmannin

    Article Snippet: VPC 23019 was obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.), SB203580, wortmannin and Y-27632 from Calbiochem (San Diego, CA, U.S.A.) and Clostridium botulinum C3 exotoxin from List Biological Laboratories Inc. (Campbell, CA, U.S.A.).

    Techniques: Migration

    Effect of the S1P receptor antagonist VPC23019 and the adenosine receptor antagonist 8-SPT on cardioprotection by 4 vs. 5 cycles of ischemic postconditioning. Isolated perfused hearts were exposed to 40 min of index ischemia and then postconditioned in the presence or absence of 1 μM VPC with either 4 cycles (4×POST) or 5 cycles (5×POST) of 15 s of reperfusion-15 s of ischemia prior to full reperfusion in the presence or absence of VPC and/or 8-SPT for 40 min. The maximum recovery of LVDP during reperfusion is reported as a percent of the preischemic value and the infarct size was determined at the end of the full 40 min of reperfusion. The controls were exposed to equilibration, index ischemia, and then reperfusion in the presence or absence of VPC and/or 8-SPT without any postconditioning treatment. The data are reported as means ± SE *These conditions (which are not significantly different from one another) are statistically different ( P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Sphingosine 1-phosphate is an important endogenous cardioprotectant released by ischemic pre- and postconditioning

    doi: 10.1152/ajpheart.00358.2009

    Figure Lengend Snippet: Effect of the S1P receptor antagonist VPC23019 and the adenosine receptor antagonist 8-SPT on cardioprotection by 4 vs. 5 cycles of ischemic postconditioning. Isolated perfused hearts were exposed to 40 min of index ischemia and then postconditioned in the presence or absence of 1 μM VPC with either 4 cycles (4×POST) or 5 cycles (5×POST) of 15 s of reperfusion-15 s of ischemia prior to full reperfusion in the presence or absence of VPC and/or 8-SPT for 40 min. The maximum recovery of LVDP during reperfusion is reported as a percent of the preischemic value and the infarct size was determined at the end of the full 40 min of reperfusion. The controls were exposed to equilibration, index ischemia, and then reperfusion in the presence or absence of VPC and/or 8-SPT without any postconditioning treatment. The data are reported as means ± SE *These conditions (which are not significantly different from one another) are statistically different ( P

    Article Snippet: VPC23019 (VPC) was obtained from Avanti Polar Lipids.

    Techniques: Single-particle Tracking, Isolation

    DH-S1P enhances endothelial barrier in an S1P1 dependent manner . Confluent EC monolayers were grown under serum-free conditions until a minimal TEER plateau was reached. In A , EC monolayers were incubated with varying concentrations of DH-S1P [111-1000 nM]. In B , EC monolayers were incubated with varying concentrations of S1P [111-1000 nM]. In C , EC monolayers were incubated with DH-S1P [1000 nM] plus and minus the S1P1 antagonist W146 or the S1P1/S1P3 antagonist VPC23019 (each at 10 μM). In D , EC monolayers were incubated with S1P [1000 nM] plus and minus the S1P1 antagonist W146 or the S1P1/S1P3 antagonist VPC23019 (each at 10 μM). Each of the TEER tracings shown is an average from three independent experiments each with two replicates per condition. Impedance values were normalized by dividing each value by the level of impedance measured just prior to the addition of effectors. As a control for A and B , monolayers were treated with 40 μg/ml delipidated albumin (Control), a concentration corresponding to the amount of BSA carrier used for the highest concentration of DH-S1P and S1P tested. As controls for C and D , ECs were treated with delipidated albumin-containing serum free medium (SFM) plus vehicle buffer.

    Journal: Lipids in Health and Disease

    Article Title: S1P, dihydro-S1P and C24:1-ceramide levels in the HDL-containing fraction of serum inversely correlate with occurrence of ischemic heart disease

    doi: 10.1186/1476-511X-10-70

    Figure Lengend Snippet: DH-S1P enhances endothelial barrier in an S1P1 dependent manner . Confluent EC monolayers were grown under serum-free conditions until a minimal TEER plateau was reached. In A , EC monolayers were incubated with varying concentrations of DH-S1P [111-1000 nM]. In B , EC monolayers were incubated with varying concentrations of S1P [111-1000 nM]. In C , EC monolayers were incubated with DH-S1P [1000 nM] plus and minus the S1P1 antagonist W146 or the S1P1/S1P3 antagonist VPC23019 (each at 10 μM). In D , EC monolayers were incubated with S1P [1000 nM] plus and minus the S1P1 antagonist W146 or the S1P1/S1P3 antagonist VPC23019 (each at 10 μM). Each of the TEER tracings shown is an average from three independent experiments each with two replicates per condition. Impedance values were normalized by dividing each value by the level of impedance measured just prior to the addition of effectors. As a control for A and B , monolayers were treated with 40 μg/ml delipidated albumin (Control), a concentration corresponding to the amount of BSA carrier used for the highest concentration of DH-S1P and S1P tested. As controls for C and D , ECs were treated with delipidated albumin-containing serum free medium (SFM) plus vehicle buffer.

    Article Snippet: For experiments evaluating the effects of the S1P1 antagonist W146 (Avanti Polar Lipids) and the S1P1/S1P3 antagonist VPC23019 (Avanti Polar Lipids) on TEER, antagonist stocks (1 mM in 5% acidified DMSO, 4 mg/ml BSA) were diluted 1:100 into the conditioned EBM at the same time that S1P or HDL was added.

    Techniques: Incubation, Concentration Assay

    S1PR4 on DC conveys ACM-dependent suppression of cytotoxicity. (A–D) T cells were co-cultured with control or ACM-primed autologous DC with or without the S1PR2/4 antagonist JTE-013 or the S1PR1/3 antagonist VPC23019. (A) Cytotoxicity induced

    Journal: European journal of immunology

    Article Title: Apoptotic tumor cells induce IL-27 release from human DC to activate regulatory T cells that express CD69 and attenuate cytotoxicity

    doi: 10.1002/eji.201142093

    Figure Lengend Snippet: S1PR4 on DC conveys ACM-dependent suppression of cytotoxicity. (A–D) T cells were co-cultured with control or ACM-primed autologous DC with or without the S1PR2/4 antagonist JTE-013 or the S1PR1/3 antagonist VPC23019. (A) Cytotoxicity induced

    Article Snippet: S1P and the S1PR1/3 inhibitor VPC23019 (1 μM) (Avanti Polar Lipids, AL, USA) were dissolved following the manufacturer’s instructions.

    Techniques: Cell Culture

    Increase in expression of sphingosine-1-phosphate receptor-1 (S1PR1) in ipsilateral leptomeningeal arteries after LtCCAO. (A) Confocal immunofluorescence double-labeling images with anti-CD31 (green) and anti-S1PR1 (red) antibodies in the ipsilateral leptomeningeal arteries 7 and 14 days after LtCCAO and in the sham-operated control. The strong S1PR1 signals (red) were detected at 7 days, but weak at 14 days after LtCCAO (bars = 10 μm). (B) Average percentage of the CD31/S1PR1 double-positive area of the total CD31 positive area after LtCCAO increased until 7 days and then decreased. (n = 6 for each group; * P

    Journal: PLoS ONE

    Article Title: Sphingosine-1-Phosphate Receptor-1 Selective Agonist Enhances Collateral Growth and Protects against Subsequent Stroke

    doi: 10.1371/journal.pone.0138029

    Figure Lengend Snippet: Increase in expression of sphingosine-1-phosphate receptor-1 (S1PR1) in ipsilateral leptomeningeal arteries after LtCCAO. (A) Confocal immunofluorescence double-labeling images with anti-CD31 (green) and anti-S1PR1 (red) antibodies in the ipsilateral leptomeningeal arteries 7 and 14 days after LtCCAO and in the sham-operated control. The strong S1PR1 signals (red) were detected at 7 days, but weak at 14 days after LtCCAO (bars = 10 μm). (B) Average percentage of the CD31/S1PR1 double-positive area of the total CD31 positive area after LtCCAO increased until 7 days and then decreased. (n = 6 for each group; * P

    Article Snippet: Fifty seven mice were randomly assigned to one of the following groups: LtCCAO and daily intraperitoneal (ip) injection for 7 days from 1hour after surgery of an S1PR1 selective agonist (SEW2871, Cayman Chemical Company, Ann Arbor, MI, 5 mg/kg/day) diluted with 0.3 mL dimethyl sulfoxide (DMSO) (SEW group, n = 15); sham surgery and daily ip injection for 7 days after surgery of SEW2871 (SEW without CCAO group, n = 6); LtCCAO and daily ip injection for 7 days after surgery of SEW2871 and an S1PR1 inverse agonist (VPC23019, Avanti Polar Lipids, Alabaster, AL, 0.5 mg/kg) diluted with 0.3 mL DMSO (SEW+VPC group, n = 8); LtCCAO and daily ip injection of DMSO (0.3 mL/day) for 7 days after surgery (vehicle group, n = 15); and no occlusion and daily ip injection of DMSO (0.3 mL/day) for 7 days after sham surgery (sham group, n = 13) ( ).

    Techniques: Expressing, Immunofluorescence, Labeling

    Proposed mechanism for the induction of inflammatory mediators by ceramide metabolites in human keratinocytes. PaCDase degrades ceramide into sphingosine in the damaged stratum corneum of the skin in patients with AD. Sphingosine is subsequently converted to sphingosine-1-phosphate (S1P) by SphK. S1P binds to S1P receptor 1 and/or 3, resulting in the production and release of TNF-α. The released TNF-α binds to TNF-α receptors, and IL-8 and endothelin-1 are produced via the activation of NF-κB.

    Journal: PLoS ONE

    Article Title: Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    doi: 10.1371/journal.pone.0089402

    Figure Lengend Snippet: Proposed mechanism for the induction of inflammatory mediators by ceramide metabolites in human keratinocytes. PaCDase degrades ceramide into sphingosine in the damaged stratum corneum of the skin in patients with AD. Sphingosine is subsequently converted to sphingosine-1-phosphate (S1P) by SphK. S1P binds to S1P receptor 1 and/or 3, resulting in the production and release of TNF-α. The released TNF-α binds to TNF-α receptors, and IL-8 and endothelin-1 are produced via the activation of NF-κB.

    Article Snippet: N-acetyl-D-erythro-phytosphingosine, S1P receptor antagonist (VPC 23019), and its negative control (TFA salt) were from Avanti Polar Lipids (Alabaster, AL, USA).

    Techniques: Produced, Activation Assay

    PaCDase and S1P activate NF-κB-dependent signal pathway. ( A ) PaCDase and S1P significantly increase phosphorylated NF-κB p65 levels. Nitrocellulose filters with Tris-buffered saline (-/-) or Tris-buffered saline plus 0.1% Triton X-100 alone (−/+) or with 1 mU/ml PaCDase (PaCD/+), 5 µM S1P (S1P/+), or 1 mU/ml PaCDase with 10 µM VPC 23019 (PaCD/VCP23019/+) were placed onto the stratum corneum and incubated for 4 h. The cells were washed and solubilized, lysates were cleared by centrifugation, and the equivalent amounts of protein of the supernatants were subjected to SDS-PAGE/immunoblotting with anti-phospho-NF-κB p65 (Ser536). To determine the amount of NF-κB p65 in each band, the membranes were re-probed with anti-NF-κB p65. The blots shown are representative of 3 independent experiments. The band intensity of phosphorylated NF-κB p65 is shown relative to that of NF-κB p65 in each lane. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. *P

    Journal: PLoS ONE

    Article Title: Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    doi: 10.1371/journal.pone.0089402

    Figure Lengend Snippet: PaCDase and S1P activate NF-κB-dependent signal pathway. ( A ) PaCDase and S1P significantly increase phosphorylated NF-κB p65 levels. Nitrocellulose filters with Tris-buffered saline (-/-) or Tris-buffered saline plus 0.1% Triton X-100 alone (−/+) or with 1 mU/ml PaCDase (PaCD/+), 5 µM S1P (S1P/+), or 1 mU/ml PaCDase with 10 µM VPC 23019 (PaCD/VCP23019/+) were placed onto the stratum corneum and incubated for 4 h. The cells were washed and solubilized, lysates were cleared by centrifugation, and the equivalent amounts of protein of the supernatants were subjected to SDS-PAGE/immunoblotting with anti-phospho-NF-κB p65 (Ser536). To determine the amount of NF-κB p65 in each band, the membranes were re-probed with anti-NF-κB p65. The blots shown are representative of 3 independent experiments. The band intensity of phosphorylated NF-κB p65 is shown relative to that of NF-κB p65 in each lane. The data are expressed as the ratio relative to control (-/-) and represent the mean±SD of 3 independent experiments. *P

    Article Snippet: N-acetyl-D-erythro-phytosphingosine, S1P receptor antagonist (VPC 23019), and its negative control (TFA salt) were from Avanti Polar Lipids (Alabaster, AL, USA).

    Techniques: Incubation, Centrifugation, SDS Page

    PaCDase-produced S1P induces endothelin-1 and IL-8 production by keratinocytes. ( A ) Involvement of SphK and S1P receptor in PaCDase-enhanced endothelin-1 and IL-8 gene expression. Nitrocellulose filters without (-) or with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum, and endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 3 independent experiments. **P

    Journal: PLoS ONE

    Article Title: Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    doi: 10.1371/journal.pone.0089402

    Figure Lengend Snippet: PaCDase-produced S1P induces endothelin-1 and IL-8 production by keratinocytes. ( A ) Involvement of SphK and S1P receptor in PaCDase-enhanced endothelin-1 and IL-8 gene expression. Nitrocellulose filters without (-) or with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed on the stratum corneum, and endothelin-1 and IL-8 mRNAs were assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 3 independent experiments. **P

    Article Snippet: N-acetyl-D-erythro-phytosphingosine, S1P receptor antagonist (VPC 23019), and its negative control (TFA salt) were from Avanti Polar Lipids (Alabaster, AL, USA).

    Techniques: Produced, Expressing, Quantitative RT-PCR

    TNF-α is expressed in all layers of PaCDase-treated keratinocytes. ( A ) In situ hybridization. Nitrocellulose filters with Tris-buffered saline alone (-/-), without or with 1 mU/ml PaCDase or 5 µM S1P in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, embedded in paraffin, and sectioned by cryostat. The sections were fixed with paraformaldehyde and incubated with biotin-labeled-RNA anti-sense and sense probes for TNF-α (x400). The data shown represent 3 independent experiments. Bar: 25 µm. ( B ) Immunohistochemical analysis. Paraformaldehyde-preserved and paraffinized 3D keratinocyte culture sections as described in panel A were incubated with rabbit anti-human TNF-α (upper panel) or with hematoxylin/ eosin (lower panel). The data shown represent 3 independent experiments. Bar: 25 µm.

    Journal: PLoS ONE

    Article Title: Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    doi: 10.1371/journal.pone.0089402

    Figure Lengend Snippet: TNF-α is expressed in all layers of PaCDase-treated keratinocytes. ( A ) In situ hybridization. Nitrocellulose filters with Tris-buffered saline alone (-/-), without or with 1 mU/ml PaCDase or 5 µM S1P in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, embedded in paraffin, and sectioned by cryostat. The sections were fixed with paraformaldehyde and incubated with biotin-labeled-RNA anti-sense and sense probes for TNF-α (x400). The data shown represent 3 independent experiments. Bar: 25 µm. ( B ) Immunohistochemical analysis. Paraformaldehyde-preserved and paraffinized 3D keratinocyte culture sections as described in panel A were incubated with rabbit anti-human TNF-α (upper panel) or with hematoxylin/ eosin (lower panel). The data shown represent 3 independent experiments. Bar: 25 µm.

    Article Snippet: N-acetyl-D-erythro-phytosphingosine, S1P receptor antagonist (VPC 23019), and its negative control (TFA salt) were from Avanti Polar Lipids (Alabaster, AL, USA).

    Techniques: In Situ Hybridization, Incubation, Labeling, Immunohistochemistry

    PaCDase enhances TNF-α gene expression via S1P and S1P receptors in 3D keratinocytes. ( A ) PaCDase induces TNF-α gene expression in 3D keratinocytes. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated), with or without 0.1% Triton X-100, were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 10 independent experiments. **P

    Journal: PLoS ONE

    Article Title: Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

    doi: 10.1371/journal.pone.0089402

    Figure Lengend Snippet: PaCDase enhances TNF-α gene expression via S1P and S1P receptors in 3D keratinocytes. ( A ) PaCDase induces TNF-α gene expression in 3D keratinocytes. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated), with or without 0.1% Triton X-100, were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 10 independent experiments. **P

    Article Snippet: N-acetyl-D-erythro-phytosphingosine, S1P receptor antagonist (VPC 23019), and its negative control (TFA salt) were from Avanti Polar Lipids (Alabaster, AL, USA).

    Techniques: Expressing, Incubation, Quantitative RT-PCR

    Sphingosine-1-phosphate (S1P)-primed neutrophils for antineutrophil cytoplasmic antibody (ANCA)-induced respiratory burst and degranulation. (A) Neutrophil respiratory burst induced by patient-derived ANCA-positive IgG was measured by conversion of dihydrorhodamine-123 (DHR-123) to rhodamine-123 in S1P-primed cells in the presence and absence of CYM50358, VPC23019 and FTY720, respectively. (B) ANCA-induced neutrophil degranulation was determined by measuring the lactoferrin concentrations in the supernatant of neutrophil degranulation reaction. CYM50358, VPC23019 and FTY720 reduced ANCA-induced lactoferrin release.

    Journal: Arthritis Research & Therapy

    Article Title: The interaction between C5a and sphingosine-1-phosphate in neutrophils for antineutrophil cytoplasmic antibody mediated activation

    doi: 10.1186/ar4604

    Figure Lengend Snippet: Sphingosine-1-phosphate (S1P)-primed neutrophils for antineutrophil cytoplasmic antibody (ANCA)-induced respiratory burst and degranulation. (A) Neutrophil respiratory burst induced by patient-derived ANCA-positive IgG was measured by conversion of dihydrorhodamine-123 (DHR-123) to rhodamine-123 in S1P-primed cells in the presence and absence of CYM50358, VPC23019 and FTY720, respectively. (B) ANCA-induced neutrophil degranulation was determined by measuring the lactoferrin concentrations in the supernatant of neutrophil degranulation reaction. CYM50358, VPC23019 and FTY720 reduced ANCA-induced lactoferrin release.

    Article Snippet: VPC23019 (Tocris, Louis, USA) is a specific antagonist for S1P receptor 1 and 3 [ ].

    Techniques: Derivative Assay

    (a) Effects of combinational pretreatment of VPC23019 (3 μM) and JTE013 (1 μM) or the vehicle (dimethyl sulfoxide, Con) on S1P infusion (8.4 nmol/min.)-induced sustained vasoconstriction in normotensive (NL) and hypertensive lungs (HL). *

    Journal: Pulmonary Circulation

    Article Title: S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs

    doi: 10.4103/2045-8932.87309

    Figure Lengend Snippet: (a) Effects of combinational pretreatment of VPC23019 (3 μM) and JTE013 (1 μM) or the vehicle (dimethyl sulfoxide, Con) on S1P infusion (8.4 nmol/min.)-induced sustained vasoconstriction in normotensive (NL) and hypertensive lungs (HL). *

    Article Snippet: [ ] Because preliminary experiments showed that bolus injections of S1P into the lung perfusate caused only transient vasoconstrictions (presumably due to rapid degradation within the pulmonary vascular bed), S1P (Enzo Life Sciences) was infused via the pulmonary artery catheter at a constant rate (8.4 nmol/ min) for 10 minutes with and without combinational pretreatment (added 15 min. prior to S1P infusion) of the dual S1P1 and 3 receptor antagonist VPC23019 (3 mM; Avanti)[ ] and the S1P2 receptor antagonist JTE013 (1 μM; Tocris)[ ] in normotensive (NL) and hypertensive lungs (HL).

    Techniques:

    The analysis of the effect of VPC23019 in the experimental asthma mouse model. OVA-sensitized mice were administered PBS or anti-CCL20 antibody (20 μg/mouse) via i.p. injection before OVA inhalation on days 21 and 22. At day 23, BALF was collected, and inflammatory cells were analyzed. Data are expressed as the mean ± SEM obtained from three mice per group (A). Lungs and BALF were collected one day after the last challenge of the OVA-sensitized mice with the aerosol containing OVA or with PBS alone. Some mice received an intraperitoneal injection of 1 μg VPC23019 in a volume of 500 μl sterile PBS 30 minutes before each OVA exposure. Lung sections were subjected to H E staining or PAS staining. Bars , 100 μm. PAS + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of PAS + epithelial cells was determined as 100 × (PAS + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (B). The total inflammatory cells in the BALF were counted. For differential inflammatory cells counting, inflammatory cells were spun down and stained with Diff-Quick. Data are expressed as the mean ± SEM obtained for three mice in each group (C). Paraffin-embedded sections of the lung were stained with the antibody against CCL20 ( brown ) CCL20 + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of CCL20 + epithelial cells was determined as 100 × (CCL20 + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (D). BEAS-2B cells were treated with S1P (1 μM) and VPC23019 (10 μM) (B) for 3 h, and CCL3, TIMP2, and IL-8 gene expressions were analyzed by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3) (E). *, P

    Journal: PLoS ONE

    Article Title: Role of S1P/S1PR3 axis in release of CCL20 from human bronchial epithelial cells

    doi: 10.1371/journal.pone.0203211

    Figure Lengend Snippet: The analysis of the effect of VPC23019 in the experimental asthma mouse model. OVA-sensitized mice were administered PBS or anti-CCL20 antibody (20 μg/mouse) via i.p. injection before OVA inhalation on days 21 and 22. At day 23, BALF was collected, and inflammatory cells were analyzed. Data are expressed as the mean ± SEM obtained from three mice per group (A). Lungs and BALF were collected one day after the last challenge of the OVA-sensitized mice with the aerosol containing OVA or with PBS alone. Some mice received an intraperitoneal injection of 1 μg VPC23019 in a volume of 500 μl sterile PBS 30 minutes before each OVA exposure. Lung sections were subjected to H E staining or PAS staining. Bars , 100 μm. PAS + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of PAS + epithelial cells was determined as 100 × (PAS + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (B). The total inflammatory cells in the BALF were counted. For differential inflammatory cells counting, inflammatory cells were spun down and stained with Diff-Quick. Data are expressed as the mean ± SEM obtained for three mice in each group (C). Paraffin-embedded sections of the lung were stained with the antibody against CCL20 ( brown ) CCL20 + bronchial epithelial cells and total epithelial cells were counted on the specimens and the percentage of CCL20 + epithelial cells was determined as 100 × (CCL20 + cell number)/(total epithelial cell number) (%). Data are expressed as the mean ± SEM (n = 3) (D). BEAS-2B cells were treated with S1P (1 μM) and VPC23019 (10 μM) (B) for 3 h, and CCL3, TIMP2, and IL-8 gene expressions were analyzed by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM (n = 3) (E). *, P

    Article Snippet: Reagents VPC23019 (also known as BMS-345541; 401480, Calbiochem, Darmstadt, Germany) and JTE23019 (Cayman, Ann Arbor, MI) were commercially obtained.

    Techniques: Mouse Assay, Injection, Staining, Diff-Quik, Quantitative RT-PCR

    Proposed scheme for S1P regulation of adipocyte proliferation and differentiation by the S1pr1/S1pr3–S1pr2 axis. JTE-013 was used as a S1pr2-specific antagonist and VPC-23019 as a S1pr1/S1pr3-specific antagonist.

    Journal: Endocrinology

    Article Title: Blockade of Sphingosine 1-Phosphate Receptor 2 Signaling Attenuates High-Fat Diet-Induced Adipocyte Hypertrophy and Systemic Glucose Intolerance in Mice

    doi: 10.1210/en.2015-1768

    Figure Lengend Snippet: Proposed scheme for S1P regulation of adipocyte proliferation and differentiation by the S1pr1/S1pr3–S1pr2 axis. JTE-013 was used as a S1pr2-specific antagonist and VPC-23019 as a S1pr1/S1pr3-specific antagonist.

    Article Snippet: Because both VPC-23019 and JTE-013 regulated differentiation of 3T3-F442A preadipocytes , these cells were employed for S1pr1 / S1pr2 knockdown experiments.

    Techniques:

    S1P and S1pr antagonists regulate preadipocyte proliferation. A, Western blot analysis of Sphk1 expression in the heart, epididymal adipocytes, and preadipocytic/differentiated 3T3-L1 cells. β-Actin was detected as loading controls. B, Representative fluorescent images of EdU (red)-labeled and Hoechst 33342 (blue)-labeled 3T3-L1 preadipocytes after a 24-hour incubation with S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM). Scale bar, 100 μm. C, Percentages of EdU-labeled (proliferative) cells/Hoechst 33342-labeled (total) cells. Over 100 Hoechst 33342-labeled cells were counted in 4 independent wells. D, WST proliferation assay of 3T3-L1 adipocytes after a 24-hour incubation with graded concentrations of S1P, VPC-23019, JTE-013, or CYM-50358. E, WST proliferation assay of 3T3-F442A adipocytes after a 24-hour incubation with S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM). F, Western blot analyses of 3T3-L1 preadipocytes using anti-Erk and anti-p-ERK antibodies. Cells were incubated with S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM) for 1 hour, and cell lysates were analyzed for Erk phosphorylation/activation. Representative images are presented. Data are mean ± SEM (n, sample numbers); *, P

    Journal: Endocrinology

    Article Title: Blockade of Sphingosine 1-Phosphate Receptor 2 Signaling Attenuates High-Fat Diet-Induced Adipocyte Hypertrophy and Systemic Glucose Intolerance in Mice

    doi: 10.1210/en.2015-1768

    Figure Lengend Snippet: S1P and S1pr antagonists regulate preadipocyte proliferation. A, Western blot analysis of Sphk1 expression in the heart, epididymal adipocytes, and preadipocytic/differentiated 3T3-L1 cells. β-Actin was detected as loading controls. B, Representative fluorescent images of EdU (red)-labeled and Hoechst 33342 (blue)-labeled 3T3-L1 preadipocytes after a 24-hour incubation with S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM). Scale bar, 100 μm. C, Percentages of EdU-labeled (proliferative) cells/Hoechst 33342-labeled (total) cells. Over 100 Hoechst 33342-labeled cells were counted in 4 independent wells. D, WST proliferation assay of 3T3-L1 adipocytes after a 24-hour incubation with graded concentrations of S1P, VPC-23019, JTE-013, or CYM-50358. E, WST proliferation assay of 3T3-F442A adipocytes after a 24-hour incubation with S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM). F, Western blot analyses of 3T3-L1 preadipocytes using anti-Erk and anti-p-ERK antibodies. Cells were incubated with S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM) for 1 hour, and cell lysates were analyzed for Erk phosphorylation/activation. Representative images are presented. Data are mean ± SEM (n, sample numbers); *, P

    Article Snippet: Because both VPC-23019 and JTE-013 regulated differentiation of 3T3-F442A preadipocytes , these cells were employed for S1pr1 / S1pr2 knockdown experiments.

    Techniques: Western Blot, Expressing, Labeling, Incubation, Proliferation Assay, Activation Assay

    S1P and S1pr antagonists regulate adipogenic differentiation of 3T3-F442A preadipocytes. When 3T3-F442A preadipocytes reached confluence, insulin (10nM) plus S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM) were added and incubated for 5 days. A, Representative images of Oil-Red O staining. B, Triglyceride (TG) contents were examined by Oil-Red O staining and normalized by genomic DNA (gDNA) contents in the cell lysates. C, Effects of S1P (10μM), VPC-23019 (10μM), and JTE-013 (10μM) on the expression of adipocyte-specific genes: Ppar γ, Fabp4 , Lpl , and Adipoq . Data are mean ± SEM (n, sample numbers); *, P

    Journal: Endocrinology

    Article Title: Blockade of Sphingosine 1-Phosphate Receptor 2 Signaling Attenuates High-Fat Diet-Induced Adipocyte Hypertrophy and Systemic Glucose Intolerance in Mice

    doi: 10.1210/en.2015-1768

    Figure Lengend Snippet: S1P and S1pr antagonists regulate adipogenic differentiation of 3T3-F442A preadipocytes. When 3T3-F442A preadipocytes reached confluence, insulin (10nM) plus S1P (10μM), VPC-23019 (10μM), JTE-013 (10μM), or CYM-50358 (1μM) were added and incubated for 5 days. A, Representative images of Oil-Red O staining. B, Triglyceride (TG) contents were examined by Oil-Red O staining and normalized by genomic DNA (gDNA) contents in the cell lysates. C, Effects of S1P (10μM), VPC-23019 (10μM), and JTE-013 (10μM) on the expression of adipocyte-specific genes: Ppar γ, Fabp4 , Lpl , and Adipoq . Data are mean ± SEM (n, sample numbers); *, P

    Article Snippet: Because both VPC-23019 and JTE-013 regulated differentiation of 3T3-F442A preadipocytes , these cells were employed for S1pr1 / S1pr2 knockdown experiments.

    Techniques: Incubation, Staining, Expressing

    COX-2 expression and PGI-2 release induced by HDL were inhibited by different S1P receptors (S1PRs) inhibitors in HUVECs. a – c Cells were washed and pretreated with PBS or the S1P1 and S1P3 inhibitor VPC23019 (10 μmol/L), or the S1P2 inhibitor JTE013 (10 μmol/L) for 1 h, and then stimulated without or with 120 μg/ml HDL for 8 h. The expression of COX-2 was assayed by Western blot analysis ( a ), relative protein expression of COX-2 was normalized to GAPDH ( b ),and the production of PGI-2 was determined by competitive ELISA ( c ). Data were mean ± SE from three separate experiments. * P

    Journal: Molecular and Cellular Biochemistry

    Article Title: High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

    doi: 10.1007/s11010-013-1941-y

    Figure Lengend Snippet: COX-2 expression and PGI-2 release induced by HDL were inhibited by different S1P receptors (S1PRs) inhibitors in HUVECs. a – c Cells were washed and pretreated with PBS or the S1P1 and S1P3 inhibitor VPC23019 (10 μmol/L), or the S1P2 inhibitor JTE013 (10 μmol/L) for 1 h, and then stimulated without or with 120 μg/ml HDL for 8 h. The expression of COX-2 was assayed by Western blot analysis ( a ), relative protein expression of COX-2 was normalized to GAPDH ( b ),and the production of PGI-2 was determined by competitive ELISA ( c ). Data were mean ± SE from three separate experiments. * P

    Article Snippet: Transfected HUVECs were serum-starved overnight in phosphate-free DMEM, metabolically labeled by incubation in medium containing [32 P]orthophosphate (70 μCi/ml) at 37 °C for 4 h, and subsequently treated with PTX (100 ng/ml, 24 h), JTE013 (10 μmol/L, 1 h), VPC23019 (10 μmol/L, 1 h), PD98059 (10 μmol/L, 1 h), GF109203X (10 μmol/L, 1 h), or their respective vehicle controls.

    Techniques: Expressing, Western Blot, Competitive ELISA