rabbit anti s1pr1  (Alomone Labs)


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    Alomone Labs rabbit anti s1pr1
    Hypothetical mechanism by which CNTF/Stat3 and <t>S1P/S1PR1</t> interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.
    Rabbit Anti S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti s1pr1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti s1pr1 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System"

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    Journal: Neural Plasticity

    doi: 10.1155/2017/6818970

    Hypothetical mechanism by which CNTF/Stat3 and S1P/S1PR1 interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.
    Figure Legend Snippet: Hypothetical mechanism by which CNTF/Stat3 and S1P/S1PR1 interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.

    Techniques Used: Activation Assay, Expressing, Translocation Assay

    S1PR1 knockdown potentiates CNTF-induced axonal regeneration. (a) Axonal regeneration was visualized on longitudinal sections of optic nerves two weeks after crush injury and 4 weeks after coinfection with ShH10.CNTF and AAV2 vectors. Axons were traced with cholera toxin β subunit (CTb) conjugated to Alexa 594 the day before tissue fixation. (b) The infection of retinal cells with ShH10.CNTF and AAV2.shRNA-S1PR1 promoted lengthy axonal regeneration in the optic nerve compared with the ShH10.CNTF/AAV2.GFP combination. (c) Quantitatively, axonal fibers were significantly more numerous between 1300 and 1800 μ m past the lesion site with ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 6 mice) than with ShH10.CNTF/AAV2.GFP ( n = 5 mice) treatments (ANOVA, ∗ p < 0.05). ShH10.CNTF/AAV2.S1PR1 did not influence axonal regeneration ( n = 6 mice). (d) The measurement of the longest axons revealed better growth distances in ShH10.CNTF-/AAV2.shRNA-S1PR1-treated animals than in mice receiving ShH10.CNTF/AAV2.GFP. Scale bars: (b) top = 200 μ m; (b) bottom = 100 μ m.
    Figure Legend Snippet: S1PR1 knockdown potentiates CNTF-induced axonal regeneration. (a) Axonal regeneration was visualized on longitudinal sections of optic nerves two weeks after crush injury and 4 weeks after coinfection with ShH10.CNTF and AAV2 vectors. Axons were traced with cholera toxin β subunit (CTb) conjugated to Alexa 594 the day before tissue fixation. (b) The infection of retinal cells with ShH10.CNTF and AAV2.shRNA-S1PR1 promoted lengthy axonal regeneration in the optic nerve compared with the ShH10.CNTF/AAV2.GFP combination. (c) Quantitatively, axonal fibers were significantly more numerous between 1300 and 1800 μ m past the lesion site with ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 6 mice) than with ShH10.CNTF/AAV2.GFP ( n = 5 mice) treatments (ANOVA, ∗ p < 0.05). ShH10.CNTF/AAV2.S1PR1 did not influence axonal regeneration ( n = 6 mice). (d) The measurement of the longest axons revealed better growth distances in ShH10.CNTF-/AAV2.shRNA-S1PR1-treated animals than in mice receiving ShH10.CNTF/AAV2.GFP. Scale bars: (b) top = 200 μ m; (b) bottom = 100 μ m.

    Techniques Used: Infection, shRNA

    Modulation of S1PR1 expression by the CNTF/Stat3 pathway in optic nerve-injured retinae. (a, b) S1PR1 expression changes were monitored 3 d after ONC in retinae infected with ShH10.CNTF or ShH10.Empty, a control vector that was deprived of cDNA sequence. ShH10 viruses preferentially infected Müller glia in the retina . P-Stat3 and S1PR1 were markedly increased by ShH10.CNTF in RGC somata identified using β 3-tubulin as a specific marker. (c) Five days after ONC, qRT-PCR measurements showed that the infection of RGCs with AAV2.Stat3 significantly increased the mRNA level of S1pr1 compared with control AAV2.GFP vector. ShH10 and AAV2 viruses were intravitreally injected 4 weeks before ONC. Three mice were analyzed/grouped. Statistics: one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; NS: not significant. Scale bar: 50 μ m.
    Figure Legend Snippet: Modulation of S1PR1 expression by the CNTF/Stat3 pathway in optic nerve-injured retinae. (a, b) S1PR1 expression changes were monitored 3 d after ONC in retinae infected with ShH10.CNTF or ShH10.Empty, a control vector that was deprived of cDNA sequence. ShH10 viruses preferentially infected Müller glia in the retina . P-Stat3 and S1PR1 were markedly increased by ShH10.CNTF in RGC somata identified using β 3-tubulin as a specific marker. (c) Five days after ONC, qRT-PCR measurements showed that the infection of RGCs with AAV2.Stat3 significantly increased the mRNA level of S1pr1 compared with control AAV2.GFP vector. ShH10 and AAV2 viruses were intravitreally injected 4 weeks before ONC. Three mice were analyzed/grouped. Statistics: one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; NS: not significant. Scale bar: 50 μ m.

    Techniques Used: Expressing, Infection, Plasmid Preparation, Sequencing, Marker, Quantitative RT-PCR, Injection

    S1PR1 knockdown alters CNTF-induced RGC survival after ONC. (a) Two weeks after ONC, surviving RGCs were observed in retinal flat-mounts after immunofluorescent staining for β 3-tubulin. Less RGCs were visible in retinae infected with AAV2.shRNA-S1PR1 and ShH10.CNTF ( n = 5 mice) than in mice injected with ShH10.CNTF/AAV2.GFP ( n = 7 mice) or ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 5 mice). (b) Quantitatively, the average number of surviving RGCs was statistically lower in whole retinae transduced with ShH10.CNTF/AAV2.shRNA-S1PR1 than in the two other groups of animals (ANOVA, ∗∗∗ p < 0.001). (c) The reduction of RGC survival caused by ShH10.CNTF/AAV2.shRNA-S1PR1 was the most pronounced in the superior quadrant of the retina (ANOVA, ∗∗∗ p < 0.001). Scale bar: 100 μ m.
    Figure Legend Snippet: S1PR1 knockdown alters CNTF-induced RGC survival after ONC. (a) Two weeks after ONC, surviving RGCs were observed in retinal flat-mounts after immunofluorescent staining for β 3-tubulin. Less RGCs were visible in retinae infected with AAV2.shRNA-S1PR1 and ShH10.CNTF ( n = 5 mice) than in mice injected with ShH10.CNTF/AAV2.GFP ( n = 7 mice) or ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 5 mice). (b) Quantitatively, the average number of surviving RGCs was statistically lower in whole retinae transduced with ShH10.CNTF/AAV2.shRNA-S1PR1 than in the two other groups of animals (ANOVA, ∗∗∗ p < 0.001). (c) The reduction of RGC survival caused by ShH10.CNTF/AAV2.shRNA-S1PR1 was the most pronounced in the superior quadrant of the retina (ANOVA, ∗∗∗ p < 0.001). Scale bar: 100 μ m.

    Techniques Used: Staining, Infection, shRNA, Injection, Transduction

    The expression of P-Stat3 is not changed by S1PR1 silencing after CNTF stimulation. The expression of P-Stat3 was assessed by western blotting in protein lysates (20 μ g) from retinae treated with ShH10 and AAV2 viruses. P-Stat3 and Stat3 blots were quantified by densitometry using the ImageJ software (NIH). The level of P-Stat3/Stat3 was not significantly different between mice treated with ShH10.CNTF/AAV2.shRNA-S1PR1 and ShH10.CNTF/AAV2.GFP. Three mice were analyzed for each group.
    Figure Legend Snippet: The expression of P-Stat3 is not changed by S1PR1 silencing after CNTF stimulation. The expression of P-Stat3 was assessed by western blotting in protein lysates (20 μ g) from retinae treated with ShH10 and AAV2 viruses. P-Stat3 and Stat3 blots were quantified by densitometry using the ImageJ software (NIH). The level of P-Stat3/Stat3 was not significantly different between mice treated with ShH10.CNTF/AAV2.shRNA-S1PR1 and ShH10.CNTF/AAV2.GFP. Three mice were analyzed for each group.

    Techniques Used: Expressing, Western Blot, Software, shRNA

    rabbit anti s1pr1  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti s1pr1
    Hypothetical mechanism by which CNTF/Stat3 and <t>S1P/S1PR1</t> interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.
    Rabbit Anti S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti s1pr1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti s1pr1 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System"

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    Journal: Neural Plasticity

    doi: 10.1155/2017/6818970

    Hypothetical mechanism by which CNTF/Stat3 and S1P/S1PR1 interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.
    Figure Legend Snippet: Hypothetical mechanism by which CNTF/Stat3 and S1P/S1PR1 interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.

    Techniques Used: Activation Assay, Expressing, Translocation Assay

    S1PR1 knockdown potentiates CNTF-induced axonal regeneration. (a) Axonal regeneration was visualized on longitudinal sections of optic nerves two weeks after crush injury and 4 weeks after coinfection with ShH10.CNTF and AAV2 vectors. Axons were traced with cholera toxin β subunit (CTb) conjugated to Alexa 594 the day before tissue fixation. (b) The infection of retinal cells with ShH10.CNTF and AAV2.shRNA-S1PR1 promoted lengthy axonal regeneration in the optic nerve compared with the ShH10.CNTF/AAV2.GFP combination. (c) Quantitatively, axonal fibers were significantly more numerous between 1300 and 1800 μ m past the lesion site with ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 6 mice) than with ShH10.CNTF/AAV2.GFP ( n = 5 mice) treatments (ANOVA, ∗ p < 0.05). ShH10.CNTF/AAV2.S1PR1 did not influence axonal regeneration ( n = 6 mice). (d) The measurement of the longest axons revealed better growth distances in ShH10.CNTF-/AAV2.shRNA-S1PR1-treated animals than in mice receiving ShH10.CNTF/AAV2.GFP. Scale bars: (b) top = 200 μ m; (b) bottom = 100 μ m.
    Figure Legend Snippet: S1PR1 knockdown potentiates CNTF-induced axonal regeneration. (a) Axonal regeneration was visualized on longitudinal sections of optic nerves two weeks after crush injury and 4 weeks after coinfection with ShH10.CNTF and AAV2 vectors. Axons were traced with cholera toxin β subunit (CTb) conjugated to Alexa 594 the day before tissue fixation. (b) The infection of retinal cells with ShH10.CNTF and AAV2.shRNA-S1PR1 promoted lengthy axonal regeneration in the optic nerve compared with the ShH10.CNTF/AAV2.GFP combination. (c) Quantitatively, axonal fibers were significantly more numerous between 1300 and 1800 μ m past the lesion site with ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 6 mice) than with ShH10.CNTF/AAV2.GFP ( n = 5 mice) treatments (ANOVA, ∗ p < 0.05). ShH10.CNTF/AAV2.S1PR1 did not influence axonal regeneration ( n = 6 mice). (d) The measurement of the longest axons revealed better growth distances in ShH10.CNTF-/AAV2.shRNA-S1PR1-treated animals than in mice receiving ShH10.CNTF/AAV2.GFP. Scale bars: (b) top = 200 μ m; (b) bottom = 100 μ m.

    Techniques Used: Infection, shRNA

    Modulation of S1PR1 expression by the CNTF/Stat3 pathway in optic nerve-injured retinae. (a, b) S1PR1 expression changes were monitored 3 d after ONC in retinae infected with ShH10.CNTF or ShH10.Empty, a control vector that was deprived of cDNA sequence. ShH10 viruses preferentially infected Müller glia in the retina . P-Stat3 and S1PR1 were markedly increased by ShH10.CNTF in RGC somata identified using β 3-tubulin as a specific marker. (c) Five days after ONC, qRT-PCR measurements showed that the infection of RGCs with AAV2.Stat3 significantly increased the mRNA level of S1pr1 compared with control AAV2.GFP vector. ShH10 and AAV2 viruses were intravitreally injected 4 weeks before ONC. Three mice were analyzed/grouped. Statistics: one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; NS: not significant. Scale bar: 50 μ m.
    Figure Legend Snippet: Modulation of S1PR1 expression by the CNTF/Stat3 pathway in optic nerve-injured retinae. (a, b) S1PR1 expression changes were monitored 3 d after ONC in retinae infected with ShH10.CNTF or ShH10.Empty, a control vector that was deprived of cDNA sequence. ShH10 viruses preferentially infected Müller glia in the retina . P-Stat3 and S1PR1 were markedly increased by ShH10.CNTF in RGC somata identified using β 3-tubulin as a specific marker. (c) Five days after ONC, qRT-PCR measurements showed that the infection of RGCs with AAV2.Stat3 significantly increased the mRNA level of S1pr1 compared with control AAV2.GFP vector. ShH10 and AAV2 viruses were intravitreally injected 4 weeks before ONC. Three mice were analyzed/grouped. Statistics: one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; NS: not significant. Scale bar: 50 μ m.

    Techniques Used: Expressing, Infection, Plasmid Preparation, Sequencing, Marker, Quantitative RT-PCR, Injection

    S1PR1 knockdown alters CNTF-induced RGC survival after ONC. (a) Two weeks after ONC, surviving RGCs were observed in retinal flat-mounts after immunofluorescent staining for β 3-tubulin. Less RGCs were visible in retinae infected with AAV2.shRNA-S1PR1 and ShH10.CNTF ( n = 5 mice) than in mice injected with ShH10.CNTF/AAV2.GFP ( n = 7 mice) or ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 5 mice). (b) Quantitatively, the average number of surviving RGCs was statistically lower in whole retinae transduced with ShH10.CNTF/AAV2.shRNA-S1PR1 than in the two other groups of animals (ANOVA, ∗∗∗ p < 0.001). (c) The reduction of RGC survival caused by ShH10.CNTF/AAV2.shRNA-S1PR1 was the most pronounced in the superior quadrant of the retina (ANOVA, ∗∗∗ p < 0.001). Scale bar: 100 μ m.
    Figure Legend Snippet: S1PR1 knockdown alters CNTF-induced RGC survival after ONC. (a) Two weeks after ONC, surviving RGCs were observed in retinal flat-mounts after immunofluorescent staining for β 3-tubulin. Less RGCs were visible in retinae infected with AAV2.shRNA-S1PR1 and ShH10.CNTF ( n = 5 mice) than in mice injected with ShH10.CNTF/AAV2.GFP ( n = 7 mice) or ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 5 mice). (b) Quantitatively, the average number of surviving RGCs was statistically lower in whole retinae transduced with ShH10.CNTF/AAV2.shRNA-S1PR1 than in the two other groups of animals (ANOVA, ∗∗∗ p < 0.001). (c) The reduction of RGC survival caused by ShH10.CNTF/AAV2.shRNA-S1PR1 was the most pronounced in the superior quadrant of the retina (ANOVA, ∗∗∗ p < 0.001). Scale bar: 100 μ m.

    Techniques Used: Staining, Infection, shRNA, Injection, Transduction

    The expression of P-Stat3 is not changed by S1PR1 silencing after CNTF stimulation. The expression of P-Stat3 was assessed by western blotting in protein lysates (20 μ g) from retinae treated with ShH10 and AAV2 viruses. P-Stat3 and Stat3 blots were quantified by densitometry using the ImageJ software (NIH). The level of P-Stat3/Stat3 was not significantly different between mice treated with ShH10.CNTF/AAV2.shRNA-S1PR1 and ShH10.CNTF/AAV2.GFP. Three mice were analyzed for each group.
    Figure Legend Snippet: The expression of P-Stat3 is not changed by S1PR1 silencing after CNTF stimulation. The expression of P-Stat3 was assessed by western blotting in protein lysates (20 μ g) from retinae treated with ShH10 and AAV2 viruses. P-Stat3 and Stat3 blots were quantified by densitometry using the ImageJ software (NIH). The level of P-Stat3/Stat3 was not significantly different between mice treated with ShH10.CNTF/AAV2.shRNA-S1PR1 and ShH10.CNTF/AAV2.GFP. Three mice were analyzed for each group.

    Techniques Used: Expressing, Western Blot, Software, shRNA

    rabbit anti s1pr1  (Alomone Labs)


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    Alomone Labs rabbit anti s1pr1
    MicroPET imaging of <t>S1PR1</t> activity in S aureus -infected mice. (a) Radiosynthesis of S1PR1-specific radiotracer, [ 18 F]TZ4877; (b) representative sagittal microPET images of [ 18 F]TZ4877 in mice. Comparing with sham mice, the tracer uptake was significantly higher in the infected mice, and the increased uptake of the tracer showed S aureus dose dependent; (c) the tracer uptake in the brain was quantified; time-activity curves showed that the tracer uptake in infected mice was significantly higher than mice without infections; (d) the average tracer uptake in the brain from 30 to 50 min of the PET scan showed a dose-dependent manner. Data represent the mean ± SEM, n = 3 for each group.
    Rabbit Anti S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti s1pr1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti s1pr1 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection"

    Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

    Journal: Molecular Imaging

    doi: 10.1155/2021/9982020

    MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Radiosynthesis of S1PR1-specific radiotracer, [ 18 F]TZ4877; (b) representative sagittal microPET images of [ 18 F]TZ4877 in mice. Comparing with sham mice, the tracer uptake was significantly higher in the infected mice, and the increased uptake of the tracer showed S aureus dose dependent; (c) the tracer uptake in the brain was quantified; time-activity curves showed that the tracer uptake in infected mice was significantly higher than mice without infections; (d) the average tracer uptake in the brain from 30 to 50 min of the PET scan showed a dose-dependent manner. Data represent the mean ± SEM, n = 3 for each group.
    Figure Legend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Radiosynthesis of S1PR1-specific radiotracer, [ 18 F]TZ4877; (b) representative sagittal microPET images of [ 18 F]TZ4877 in mice. Comparing with sham mice, the tracer uptake was significantly higher in the infected mice, and the increased uptake of the tracer showed S aureus dose dependent; (c) the tracer uptake in the brain was quantified; time-activity curves showed that the tracer uptake in infected mice was significantly higher than mice without infections; (d) the average tracer uptake in the brain from 30 to 50 min of the PET scan showed a dose-dependent manner. Data represent the mean ± SEM, n = 3 for each group.

    Techniques Used: Imaging, Activity Assay, Infection

    Biodistribution (%ID/g, mean ± SEM) of  S1PR1-specific  [ 18 F]TZ4877 in Balb/c mice ( n = 4).
    Figure Legend Snippet: Biodistribution (%ID/g, mean ± SEM) of S1PR1-specific [ 18 F]TZ4877 in Balb/c mice ( n = 4).

    Techniques Used: Mouse Assay

    Biodistribution of  S1PR1-specific  [ 18 F]TZ4877 in sham, infected, and infected with treatments mice ( n = 4).
    Figure Legend Snippet: Biodistribution of S1PR1-specific [ 18 F]TZ4877 in sham, infected, and infected with treatments mice ( n = 4).

    Techniques Used: Infection, Mouse Assay

    MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Representative sagittal microPET images of [ 18 F]TZ4877 in the hind limb of mice. The tracer uptake was relatively low in the hind limb muscle with a SUV of ~1.5 in sham mice. Comparing with sham mice, the tracer uptake was significantly higher in the hind limb of infected mice; (b) time-activity curves showed that the tracer uptake in infected mice was significantly higher than sham mice; (c) the average tracer uptake in the hind limb muscle from 30 to 50 min of the PET scan showed a ~39% increase of SUV in infected mice with a P value of 0.0082. Data represent the mean ± SEM, n = 3 for each group.
    Figure Legend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Representative sagittal microPET images of [ 18 F]TZ4877 in the hind limb of mice. The tracer uptake was relatively low in the hind limb muscle with a SUV of ~1.5 in sham mice. Comparing with sham mice, the tracer uptake was significantly higher in the hind limb of infected mice; (b) time-activity curves showed that the tracer uptake in infected mice was significantly higher than sham mice; (c) the average tracer uptake in the hind limb muscle from 30 to 50 min of the PET scan showed a ~39% increase of SUV in infected mice with a P value of 0.0082. Data represent the mean ± SEM, n = 3 for each group.

    Techniques Used: Imaging, Activity Assay, Infection

    PET measurements of  S1PR1-specific  [ 18 F]TZ4877 in S aureus -infected and sham mice.
    Figure Legend Snippet: PET measurements of S1PR1-specific [ 18 F]TZ4877 in S aureus -infected and sham mice.

    Techniques Used: Infection

    Immunohistochemistry analysis of S1PR1 in hind limb muscle of sham and S aureus -infected mice. S1PR1 was significantly upregulated in the muscle of infected mice (red arrow) comparing with sham mice (green arrow), scale bar = 100 μ m.
    Figure Legend Snippet: Immunohistochemistry analysis of S1PR1 in hind limb muscle of sham and S aureus -infected mice. S1PR1 was significantly upregulated in the muscle of infected mice (red arrow) comparing with sham mice (green arrow), scale bar = 100 μ m.

    Techniques Used: Immunohistochemistry, Infection

    anti s1pr1 antibody  (Alomone Labs)


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    Alomone Labs anti s1pr1 antibody
    Immunohistochemistry (IHC) of <t>S1PR1</t> in postmortem DLPFC tissues from the representative normal control and schizophrenia Type 1 and Type 2.
    Anti S1pr1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti s1pr1 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti s1pr1 antibody - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2"

    Article Title: Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2

    Journal: Frontiers in Psychiatry

    doi: 10.3389/fpsyt.2022.827981

    Immunohistochemistry (IHC) of S1PR1 in postmortem DLPFC tissues from the representative normal control and schizophrenia Type 1 and Type 2.
    Figure Legend Snippet: Immunohistochemistry (IHC) of S1PR1 in postmortem DLPFC tissues from the representative normal control and schizophrenia Type 1 and Type 2.

    Techniques Used: Immunohistochemistry

    Representative images of [ 3 H]CS1P1 autoradiograph, S1PR1 immunostaining, and Hematoxylin and eosin (H&E) staining in postmortem human DLPFC tissues. The distribution of [ 3 H]CS1P1 matched well with anti-S1PR1 antibody, and was mainly located in the gray matter regions as indicated in the H&E staining.
    Figure Legend Snippet: Representative images of [ 3 H]CS1P1 autoradiograph, S1PR1 immunostaining, and Hematoxylin and eosin (H&E) staining in postmortem human DLPFC tissues. The distribution of [ 3 H]CS1P1 matched well with anti-S1PR1 antibody, and was mainly located in the gray matter regions as indicated in the H&E staining.

    Techniques Used: Autoradiography, Immunostaining, Staining

    Autoradiography images of S1PR1 using [ 3 H]CS1P1 in postmortem DLPFC tissues from representative normal control, schizophrenia Type 1, and schizophrenia Type 2. In general, [ 3 H]CS1P1 was higher in Type 2 schizophrenia subjects compared with normal control and Type 1 schizophrenia subjects.
    Figure Legend Snippet: Autoradiography images of S1PR1 using [ 3 H]CS1P1 in postmortem DLPFC tissues from representative normal control, schizophrenia Type 1, and schizophrenia Type 2. In general, [ 3 H]CS1P1 was higher in Type 2 schizophrenia subjects compared with normal control and Type 1 schizophrenia subjects.

    Techniques Used: Autoradiography

    ARG S1PR1 intensity expression (fmol/mg) triplicate measures (M1, M2, and M3) in the DLPFC from normal controls, schizophrenia Type 1, and schizophrenia Type 2.
    Figure Legend Snippet: ARG S1PR1 intensity expression (fmol/mg) triplicate measures (M1, M2, and M3) in the DLPFC from normal controls, schizophrenia Type 1, and schizophrenia Type 2.

    Techniques Used: Expressing

    ARG S1PR1 intensity expression (in fmol/mg) comparison between normal controls, schizophrenia Type 1, and schizophrenia Type 2 (* p < 0.05).
    Figure Legend Snippet: ARG S1PR1 intensity expression (in fmol/mg) comparison between normal controls, schizophrenia Type 1, and schizophrenia Type 2 (* p < 0.05).

    Techniques Used: Expressing

    anti s1pr1 antibody  (Alomone Labs)


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    Alomone Labs anti s1pr1 antibody
    Immunohistochemistry of <t>S1PR1</t> in control and schizophrenia DLPFC.
    Anti S1pr1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Differential sphingosine-1-phosphate receptor-1 (S1PR1) protein expressions in the dorsolateral prefrontal cortex between schizophrenia Type 1 and Type 2"

    Article Title: Differential sphingosine-1-phosphate receptor-1 (S1PR1) protein expressions in the dorsolateral prefrontal cortex between schizophrenia Type 1 and Type 2

    Journal: bioRxiv

    doi: 10.1101/2021.05.15.444302

    Immunohistochemistry of S1PR1 in control and schizophrenia DLPFC.
    Figure Legend Snippet: Immunohistochemistry of S1PR1 in control and schizophrenia DLPFC.

    Techniques Used: Immunohistochemistry

    Representative images of autoradiograph analysis of S1PR1 using S1PR1 specific [ 3 H]CS1P1 in control and schizophrenia DLPFC.
    Figure Legend Snippet: Representative images of autoradiograph analysis of S1PR1 using S1PR1 specific [ 3 H]CS1P1 in control and schizophrenia DLPFC.

    Techniques Used: Autoradiography

    Autoradiograph analysis of S1PR1 using S1PR1 specific [ 3 H]CS1P1 in control and schizophrenia DLPFC. A) Comparison among control, schizophrenia Type 1 and schizophrenia Type 2 (* represents p < 0.05), and B) Comparison between control and schizophrenia (SZ) (Type 1 and Type 2 combined).
    Figure Legend Snippet: Autoradiograph analysis of S1PR1 using S1PR1 specific [ 3 H]CS1P1 in control and schizophrenia DLPFC. A) Comparison among control, schizophrenia Type 1 and schizophrenia Type 2 (* represents p < 0.05), and B) Comparison between control and schizophrenia (SZ) (Type 1 and Type 2 combined).

    Techniques Used: Autoradiography

    s1pr1  (Alomone Labs)


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    Alomone Labs s1pr1
    OCTA images under influence of pro-and <t>anti-S1PR1</t> agent. (Note that all OCTA images shown above may contain artifact from OCTA itself). A: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the no PC model. Upper row: sham model (no PC and no drug administration). Middle row: sham + SEW model (no PC and SEW administration). Lower row: sham + VPC model (no PC and VPC administration). The appearance of the retinal blood vessels did not change, and no collateral vessel formation occurred in the sham model, sham + SEW administration, and sham + VPC administration groups (images show the deep capillary bed of the retina). B: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the RVO model. Upper row: normal RVO model (no drug administration). Middle row: RVO + SEW (SEW-administration). Lower row: RVO + VPC (VPC-administration). The number of collateral vessels in the RVO + SEW group was significantly greater than in the normal RVO group, and RVO + VPC group (both, p < 0.0001). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although this difference was not statistically significant (p = 0.1427). Right schema: the red cross shows the occlusion point. Black radial lines represent the retinal vessels. The blue tortured line represents the newly formed collateral vessels. As illustrated in the right schema, the RVO + SEW group tended to have more collateral vessels than the normal RVO group, and the RVO + VPC group tended to have fewer collateral vessels than the normal RVO group.
    S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation"

    Article Title: Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2021.e05902

    OCTA images under influence of pro-and anti-S1PR1 agent. (Note that all OCTA images shown above may contain artifact from OCTA itself). A: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the no PC model. Upper row: sham model (no PC and no drug administration). Middle row: sham + SEW model (no PC and SEW administration). Lower row: sham + VPC model (no PC and VPC administration). The appearance of the retinal blood vessels did not change, and no collateral vessel formation occurred in the sham model, sham + SEW administration, and sham + VPC administration groups (images show the deep capillary bed of the retina). B: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the RVO model. Upper row: normal RVO model (no drug administration). Middle row: RVO + SEW (SEW-administration). Lower row: RVO + VPC (VPC-administration). The number of collateral vessels in the RVO + SEW group was significantly greater than in the normal RVO group, and RVO + VPC group (both, p < 0.0001). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although this difference was not statistically significant (p = 0.1427). Right schema: the red cross shows the occlusion point. Black radial lines represent the retinal vessels. The blue tortured line represents the newly formed collateral vessels. As illustrated in the right schema, the RVO + SEW group tended to have more collateral vessels than the normal RVO group, and the RVO + VPC group tended to have fewer collateral vessels than the normal RVO group.
    Figure Legend Snippet: OCTA images under influence of pro-and anti-S1PR1 agent. (Note that all OCTA images shown above may contain artifact from OCTA itself). A: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the no PC model. Upper row: sham model (no PC and no drug administration). Middle row: sham + SEW model (no PC and SEW administration). Lower row: sham + VPC model (no PC and VPC administration). The appearance of the retinal blood vessels did not change, and no collateral vessel formation occurred in the sham model, sham + SEW administration, and sham + VPC administration groups (images show the deep capillary bed of the retina). B: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the RVO model. Upper row: normal RVO model (no drug administration). Middle row: RVO + SEW (SEW-administration). Lower row: RVO + VPC (VPC-administration). The number of collateral vessels in the RVO + SEW group was significantly greater than in the normal RVO group, and RVO + VPC group (both, p < 0.0001). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although this difference was not statistically significant (p = 0.1427). Right schema: the red cross shows the occlusion point. Black radial lines represent the retinal vessels. The blue tortured line represents the newly formed collateral vessels. As illustrated in the right schema, the RVO + SEW group tended to have more collateral vessels than the normal RVO group, and the RVO + VPC group tended to have fewer collateral vessels than the normal RVO group.

    Techniques Used: Imaging

    A: Average number of collateral vessels per eye. The average number of collateral vessels per eye was 1.66 in the normal RVO group, 4.11 in the RVO + SEW group, and 0.71 in the RVO + VPC group. The number of collateral vessels differed among the normal RVO group, RVO + SEW group, and RVO + VPC group (p < 0.0001). In addition, the number of collateral vessels in the RVO + SEW group was also significantly higher than in the normal RVO group and RVO + VPC group (p < 0.0001 for both groups). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although the difference was not statistically significant (p = 0.1427). B: RT-PCR for S1PR1 expression in the whole retina. Differences in the mean levels of S1PR1 expression were observed between the control group (no laser irradiation and no drug administration), and at time points 6 h, 12 h, 24 h, and 3 days after laser irradiation (p < 0.0001). The levels of S1PR1 expression 6 h (p < 0.0001) and 12 h (p = 0.0023) after laser irradiation were significantly higher than the sham, and these levels then gradually decreased and did not increase thereafter. When comparing S1PR1 expression levels between 6 h and 12 h post-laser irradiation, the expression at 6 h was significantly greater than at 12 h (p < 0.0001). The expression of S1PR1 mRNA peaked by about 6 h after blood vessel occlusion, and was significantly greater than control levels.
    Figure Legend Snippet: A: Average number of collateral vessels per eye. The average number of collateral vessels per eye was 1.66 in the normal RVO group, 4.11 in the RVO + SEW group, and 0.71 in the RVO + VPC group. The number of collateral vessels differed among the normal RVO group, RVO + SEW group, and RVO + VPC group (p < 0.0001). In addition, the number of collateral vessels in the RVO + SEW group was also significantly higher than in the normal RVO group and RVO + VPC group (p < 0.0001 for both groups). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although the difference was not statistically significant (p = 0.1427). B: RT-PCR for S1PR1 expression in the whole retina. Differences in the mean levels of S1PR1 expression were observed between the control group (no laser irradiation and no drug administration), and at time points 6 h, 12 h, 24 h, and 3 days after laser irradiation (p < 0.0001). The levels of S1PR1 expression 6 h (p < 0.0001) and 12 h (p = 0.0023) after laser irradiation were significantly higher than the sham, and these levels then gradually decreased and did not increase thereafter. When comparing S1PR1 expression levels between 6 h and 12 h post-laser irradiation, the expression at 6 h was significantly greater than at 12 h (p < 0.0001). The expression of S1PR1 mRNA peaked by about 6 h after blood vessel occlusion, and was significantly greater than control levels.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Irradiation

    Immunofluorescence imaging of retinal flat mounts. a: Immunohistochemical staining for CD31 at day 1 post-PC. b: Immunohistochemical staining for S1PR1 at day 1 post-PC. c: Immunohistochemical staining for CD31 at day 3 post-PC. d: Immunohistochemical staining for S1PR1 at day 3 post-PC. White oval: occluded vein. On day 1 post-PC, S1PR1 staining occurred along the laser-occluded blood vessel, but was not present along other blood vessels. There was no prominent staining for S1PR1 along the occluded blood vessels after the first day post-PC.
    Figure Legend Snippet: Immunofluorescence imaging of retinal flat mounts. a: Immunohistochemical staining for CD31 at day 1 post-PC. b: Immunohistochemical staining for S1PR1 at day 1 post-PC. c: Immunohistochemical staining for CD31 at day 3 post-PC. d: Immunohistochemical staining for S1PR1 at day 3 post-PC. White oval: occluded vein. On day 1 post-PC, S1PR1 staining occurred along the laser-occluded blood vessel, but was not present along other blood vessels. There was no prominent staining for S1PR1 along the occluded blood vessels after the first day post-PC.

    Techniques Used: Immunofluorescence, Imaging, Immunohistochemical staining, Staining

    anti sphingosine 1 phosphate receptor 1 extracellular  (Alomone Labs)


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    Alomone Labs anti sphingosine 1 phosphate receptor 1 extracellular
    (A and B) After 24 h post transfection of HPAECs with either scrambled siRNA (siScr) or <t>S1PR1</t> siRNA (siS1PR1), these cells were transduced with control vector or hemagglutinin (HA)-tagged S1PR1 cDNA to restore S1PR1 expression. Cells were stimulated with VEGF (50 ng) with or without S1P (1 μM), and network formation was assessed at 16 h by light microscopy (20× and 40×). (A) shows a representative image taken at 40× magnification, whereas (B) shows quantitation of the endothelial polygonal areas formed using 20× magnification. Bars, 50 μm (A). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to un-stimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (C) HPAECs processed as described in (B) were seeded on the top surface of Matrigel-coated polycarbonate membrane inserts of Boyden chamber. Cells were incubated with basal media along with indicated ligands (50 ng VEGF or 1 μM S1P) placed outside the chambers. After 16 h, ECs migrating to the bottom surface of the insert were fixed and nuclei were stained with DAPI, and their migration was determined using confocal microscope (20×). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to unstimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (D) Tumor growth in S1pr1 fl/fl versus EC-S1pr1 −/− mice following subcutaneous injection of 0.25 × 10 6 Lewis lung cell carcinoma (LLC) cells. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to S1pr1 fl/fl mice. Data expressed as mean ± SEM. (E) Tumor weight post day 21 of LLC cell injection in indicated strains of mice. Inset: a representative image of tumor from each strain. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 compared to S1pr1 fl/fl tumors. (F–H) S1pr1 fl/fl and EC-S1pr1 −/− tumor-associated ECs (TAECs) were isolated using FACS and cultured, and network formation and migration were assessed as described in (A)–(C). (F) shows the representative images of network formation; (G) shows the quantification of number of branch points formed; and (H) shows the quantification of the migrated cells per field. Bars, 50 μm. Image representative of multiple samples from different mice (F). Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs (G and H). All data represented were repeated multiple times and are expressed as mean ± SEM.
    Anti Sphingosine 1 Phosphate Receptor 1 Extracellular, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling"

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.11.036

    (A and B) After 24 h post transfection of HPAECs with either scrambled siRNA (siScr) or S1PR1 siRNA (siS1PR1), these cells were transduced with control vector or hemagglutinin (HA)-tagged S1PR1 cDNA to restore S1PR1 expression. Cells were stimulated with VEGF (50 ng) with or without S1P (1 μM), and network formation was assessed at 16 h by light microscopy (20× and 40×). (A) shows a representative image taken at 40× magnification, whereas (B) shows quantitation of the endothelial polygonal areas formed using 20× magnification. Bars, 50 μm (A). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to un-stimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (C) HPAECs processed as described in (B) were seeded on the top surface of Matrigel-coated polycarbonate membrane inserts of Boyden chamber. Cells were incubated with basal media along with indicated ligands (50 ng VEGF or 1 μM S1P) placed outside the chambers. After 16 h, ECs migrating to the bottom surface of the insert were fixed and nuclei were stained with DAPI, and their migration was determined using confocal microscope (20×). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to unstimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (D) Tumor growth in S1pr1 fl/fl versus EC-S1pr1 −/− mice following subcutaneous injection of 0.25 × 10 6 Lewis lung cell carcinoma (LLC) cells. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to S1pr1 fl/fl mice. Data expressed as mean ± SEM. (E) Tumor weight post day 21 of LLC cell injection in indicated strains of mice. Inset: a representative image of tumor from each strain. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 compared to S1pr1 fl/fl tumors. (F–H) S1pr1 fl/fl and EC-S1pr1 −/− tumor-associated ECs (TAECs) were isolated using FACS and cultured, and network formation and migration were assessed as described in (A)–(C). (F) shows the representative images of network formation; (G) shows the quantification of number of branch points formed; and (H) shows the quantification of the migrated cells per field. Bars, 50 μm. Image representative of multiple samples from different mice (F). Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs (G and H). All data represented were repeated multiple times and are expressed as mean ± SEM.
    Figure Legend Snippet: (A and B) After 24 h post transfection of HPAECs with either scrambled siRNA (siScr) or S1PR1 siRNA (siS1PR1), these cells were transduced with control vector or hemagglutinin (HA)-tagged S1PR1 cDNA to restore S1PR1 expression. Cells were stimulated with VEGF (50 ng) with or without S1P (1 μM), and network formation was assessed at 16 h by light microscopy (20× and 40×). (A) shows a representative image taken at 40× magnification, whereas (B) shows quantitation of the endothelial polygonal areas formed using 20× magnification. Bars, 50 μm (A). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to un-stimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (C) HPAECs processed as described in (B) were seeded on the top surface of Matrigel-coated polycarbonate membrane inserts of Boyden chamber. Cells were incubated with basal media along with indicated ligands (50 ng VEGF or 1 μM S1P) placed outside the chambers. After 16 h, ECs migrating to the bottom surface of the insert were fixed and nuclei were stained with DAPI, and their migration was determined using confocal microscope (20×). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to unstimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (D) Tumor growth in S1pr1 fl/fl versus EC-S1pr1 −/− mice following subcutaneous injection of 0.25 × 10 6 Lewis lung cell carcinoma (LLC) cells. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to S1pr1 fl/fl mice. Data expressed as mean ± SEM. (E) Tumor weight post day 21 of LLC cell injection in indicated strains of mice. Inset: a representative image of tumor from each strain. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 compared to S1pr1 fl/fl tumors. (F–H) S1pr1 fl/fl and EC-S1pr1 −/− tumor-associated ECs (TAECs) were isolated using FACS and cultured, and network formation and migration were assessed as described in (A)–(C). (F) shows the representative images of network formation; (G) shows the quantification of number of branch points formed; and (H) shows the quantification of the migrated cells per field. Bars, 50 μm. Image representative of multiple samples from different mice (F). Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs (G and H). All data represented were repeated multiple times and are expressed as mean ± SEM.

    Techniques Used: Transfection, Transduction, Plasmid Preparation, Expressing, Light Microscopy, Quantitation Assay, Two Tailed Test, Incubation, Staining, Migration, Microscopy, Injection, Isolation, Cell Culture

    (A) Schematic showing generation of mice for lineage tracing ECs using Floxed or S1PR1 fl / scl-creERT (5′ endothelial-cell-specific Scl-Cre ERT promotor) and Rosa26-tdTomato mice line. Tamoxifen injection induces Cre activity in the lox-P flanked locus, resulting in S1PR1 deletion in endothelial cells at stop sequence, turning these cells and their progeny to be labeled by tdTomato. (B and C) Indicated mice were injected with LLC cells as described in , and at 21 days, animals were injected with FITC-IB4 (intravenous [i.v.], 50 μg/100 μL) 4 h prior to sacrificing. Tumors were excised, stained with DAPI, and analyzed for tdTomato co-localization with IB4-positive vessels by using confocal microscope. (A) shows a representative image and (B) shows quantitation of vessel density. Bars, 100 μm. n = 5 mice per group. Paired t test, two-tailed, ***p < 0.001 relative to tdTomato-EC mice. (D and E) Indicated mice were treated either with VEGFR2 inhibitor (SU5416, 25 mg/kg) or S1PR1 inhibitor (R(W)146, 100 mg/kg) or a combination of both compounds. Mice received 8 doses of each compound, every 48 h beginning day 5 post LLC injection. Tumor volume was assessed using calipers every 48 h. n = 5 mice per group. One-way ANOVA, p < 0.05 (D). Post hoc Tukey’s test, ***p < 0.01 compared to R(W)146 compound, SU5416, and the combination of R(W) 146 and SU5416 injected group (D). One-way ANOVA, p > 0.05 (E). (F) TAECs seeded on poly-carbonate membrane were left untreated or treated either with SU5416 (100 μM) or R(W)146 (100 μM) or both for 2 h, and migration of tumor ECs was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to untreated S1PR1 + TAECs. #p < 0.05 compared to R(W)146-treated S1PR1 + TAECs. NS, not significantly different. All data expressed as mean ± SEM.
    Figure Legend Snippet: (A) Schematic showing generation of mice for lineage tracing ECs using Floxed or S1PR1 fl / scl-creERT (5′ endothelial-cell-specific Scl-Cre ERT promotor) and Rosa26-tdTomato mice line. Tamoxifen injection induces Cre activity in the lox-P flanked locus, resulting in S1PR1 deletion in endothelial cells at stop sequence, turning these cells and their progeny to be labeled by tdTomato. (B and C) Indicated mice were injected with LLC cells as described in , and at 21 days, animals were injected with FITC-IB4 (intravenous [i.v.], 50 μg/100 μL) 4 h prior to sacrificing. Tumors were excised, stained with DAPI, and analyzed for tdTomato co-localization with IB4-positive vessels by using confocal microscope. (A) shows a representative image and (B) shows quantitation of vessel density. Bars, 100 μm. n = 5 mice per group. Paired t test, two-tailed, ***p < 0.001 relative to tdTomato-EC mice. (D and E) Indicated mice were treated either with VEGFR2 inhibitor (SU5416, 25 mg/kg) or S1PR1 inhibitor (R(W)146, 100 mg/kg) or a combination of both compounds. Mice received 8 doses of each compound, every 48 h beginning day 5 post LLC injection. Tumor volume was assessed using calipers every 48 h. n = 5 mice per group. One-way ANOVA, p < 0.05 (D). Post hoc Tukey’s test, ***p < 0.01 compared to R(W)146 compound, SU5416, and the combination of R(W) 146 and SU5416 injected group (D). One-way ANOVA, p > 0.05 (E). (F) TAECs seeded on poly-carbonate membrane were left untreated or treated either with SU5416 (100 μM) or R(W)146 (100 μM) or both for 2 h, and migration of tumor ECs was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to untreated S1PR1 + TAECs. #p < 0.05 compared to R(W)146-treated S1PR1 + TAECs. NS, not significantly different. All data expressed as mean ± SEM.

    Techniques Used: Injection, Activity Assay, Sequencing, Labeling, Staining, Microscopy, Quantitation Assay, Two Tailed Test, Migration

    (A) Schematic showing generation of the S1PR1-GFP signaling mice . In the S1pr1 knock-in signaling mouse, S1PR1 is fused with two fusion proteins, namely, a tetracycline-regulated transactivator (tTA) and tobacco etch virus (TEV) protease along with β-arrestin. The S1pr1 knock-in signaling mouse is bred with H2B-GFP mouse to create the S1pr1-GFP signaling mouse where GFP expression reports for S1PR1 activity. (B and C) LLC cells were injected into H2B-GFP and S1PR1-GFP mice as described in . At day 16, tumors from these mice were harvested, sectioned, and stained with CD31 antibody to assess vessel density. (B) shows a representative image and (C) shows quantification of GFP+ vessel density based on CD31 staining. GFP signal serves as a proxy for S1P released by tumor cells and thereby the activation of S1PR1 in ECs. Note, a 5-fold magnified image of marked area in extreme right (B) shows S1PR1 activity in (GFP+) ECs (CD31+). Bar, 100 μm. Inset, indicates the area that was selected for magnification (5×) and presented at the right panel. n = 5 mice per group. Paired Student’s t test, ***p < 0.001 compared with H2B-GFP control. (D) qPCR of indicated genes in TAECs using specific primers. GAPDH expression was used as the internal control. One-way ANOVA for all genes. SPHK1, p < 0.05; SPHK2, p > 0.05; VEGF, p < 0.05; SPNS2, p < 0.05; GAPDH, p > 0.05. Paired Student’s t test, *p < 0.05 compared with LLCs. (E) LLCs were transfected with either scrambled siRNA (siScr) or Sphk1 (siSphk1) or Sphk2 (siSphk2) siRNA for 72 h. S1P concentrations were measured in indicated cells by using ELISA. One-way ANOVA, p < 0.01. Paired t test, two-tailed, ***p < 0.001 relative to siScr. **p < 0.01 relative to siScr. All data are representative of mean ± SEM from three individual experiments.
    Figure Legend Snippet: (A) Schematic showing generation of the S1PR1-GFP signaling mice . In the S1pr1 knock-in signaling mouse, S1PR1 is fused with two fusion proteins, namely, a tetracycline-regulated transactivator (tTA) and tobacco etch virus (TEV) protease along with β-arrestin. The S1pr1 knock-in signaling mouse is bred with H2B-GFP mouse to create the S1pr1-GFP signaling mouse where GFP expression reports for S1PR1 activity. (B and C) LLC cells were injected into H2B-GFP and S1PR1-GFP mice as described in . At day 16, tumors from these mice were harvested, sectioned, and stained with CD31 antibody to assess vessel density. (B) shows a representative image and (C) shows quantification of GFP+ vessel density based on CD31 staining. GFP signal serves as a proxy for S1P released by tumor cells and thereby the activation of S1PR1 in ECs. Note, a 5-fold magnified image of marked area in extreme right (B) shows S1PR1 activity in (GFP+) ECs (CD31+). Bar, 100 μm. Inset, indicates the area that was selected for magnification (5×) and presented at the right panel. n = 5 mice per group. Paired Student’s t test, ***p < 0.001 compared with H2B-GFP control. (D) qPCR of indicated genes in TAECs using specific primers. GAPDH expression was used as the internal control. One-way ANOVA for all genes. SPHK1, p < 0.05; SPHK2, p > 0.05; VEGF, p < 0.05; SPNS2, p < 0.05; GAPDH, p > 0.05. Paired Student’s t test, *p < 0.05 compared with LLCs. (E) LLCs were transfected with either scrambled siRNA (siScr) or Sphk1 (siSphk1) or Sphk2 (siSphk2) siRNA for 72 h. S1P concentrations were measured in indicated cells by using ELISA. One-way ANOVA, p < 0.01. Paired t test, two-tailed, ***p < 0.001 relative to siScr. **p < 0.01 relative to siScr. All data are representative of mean ± SEM from three individual experiments.

    Techniques Used: Knock-In, Expressing, Activity Assay, Injection, Staining, Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    (A and B) FACS analysis of TAECs stained with anti-S1PR1, anti-VEGFR2, or control IgG antibodies and appropriate fluorescent-tagged secondary antibodies without permeabilization. Representative FACS profiles are presented in (A), and (B) shows quantitation of S1PR1- and VEGFR2-positive cells. Experiments were repeated three times. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (B). (C and D) Analysis of S1PR1 and VEGFR2 expression in S1pr1 +/+ and S1pr1 −/− TAECs by using confocal analysis. After fixing, TAECs were permeabilized and stained with indicated antibodies, followed by Alexa fluor-labeled secondary antibodies. Images were acquired using a confocal microscope. Bar, 10 μm. (C) shows images representative of experiments that were conducted multiple times, and in (D), the quantification of surface expression of VEGFR2 normalized to the expression of total VEGFR2 is represented by considering 20 cells. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (D). (E and F) HEK cells co-transfected with FLAG-VEGFR2 cDNA along with empty vector or HA-S1PR1 cDNA were biotinylated and then stimulated with 50 ng VEGF for indicated times. Total and cell surface expression of VEGFR2 and S1PR1 was determined using anti-VEGFR2 and anti-S1PR1 antibodies. A representative immunoblot is shown in (E), and (F) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, *p < 0.05 relative to cells transfected with VEGFR2 cDNA alone post without or 5 min VEGF stimulation and relative to cells transfected with VEGFR2 plus S1PR1 cDNA post 0–60 min of VEGF stimulation (E). UD, undetectable. Immunoblot is representative of three individual experiments. (G) At day 21, LLC tumor-bearing tdTomato-EC and tdTomato-EC-S1pr1 −/− mice were injected with FITC-IB4 (50 μg/100 μL, i.v). Tumors were harvested after 4 h, sectioned, and stained with anti-VEGFR2 antibody and DAPI. Tumor vessels were visualized using a confocal microscope. Right panel shows a 5-fold magnified area of marked tdTomato-positive vessel showing co-localizing of VEGFR2 with IB4. Bar, 50 μm. n = 5mice/group. All data expressed as mean ± SEM.
    Figure Legend Snippet: (A and B) FACS analysis of TAECs stained with anti-S1PR1, anti-VEGFR2, or control IgG antibodies and appropriate fluorescent-tagged secondary antibodies without permeabilization. Representative FACS profiles are presented in (A), and (B) shows quantitation of S1PR1- and VEGFR2-positive cells. Experiments were repeated three times. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (B). (C and D) Analysis of S1PR1 and VEGFR2 expression in S1pr1 +/+ and S1pr1 −/− TAECs by using confocal analysis. After fixing, TAECs were permeabilized and stained with indicated antibodies, followed by Alexa fluor-labeled secondary antibodies. Images were acquired using a confocal microscope. Bar, 10 μm. (C) shows images representative of experiments that were conducted multiple times, and in (D), the quantification of surface expression of VEGFR2 normalized to the expression of total VEGFR2 is represented by considering 20 cells. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (D). (E and F) HEK cells co-transfected with FLAG-VEGFR2 cDNA along with empty vector or HA-S1PR1 cDNA were biotinylated and then stimulated with 50 ng VEGF for indicated times. Total and cell surface expression of VEGFR2 and S1PR1 was determined using anti-VEGFR2 and anti-S1PR1 antibodies. A representative immunoblot is shown in (E), and (F) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, *p < 0.05 relative to cells transfected with VEGFR2 cDNA alone post without or 5 min VEGF stimulation and relative to cells transfected with VEGFR2 plus S1PR1 cDNA post 0–60 min of VEGF stimulation (E). UD, undetectable. Immunoblot is representative of three individual experiments. (G) At day 21, LLC tumor-bearing tdTomato-EC and tdTomato-EC-S1pr1 −/− mice were injected with FITC-IB4 (50 μg/100 μL, i.v). Tumors were harvested after 4 h, sectioned, and stained with anti-VEGFR2 antibody and DAPI. Tumor vessels were visualized using a confocal microscope. Right panel shows a 5-fold magnified area of marked tdTomato-positive vessel showing co-localizing of VEGFR2 with IB4. Bar, 50 μm. n = 5mice/group. All data expressed as mean ± SEM.

    Techniques Used: Staining, Quantitation Assay, Two Tailed Test, Expressing, Labeling, Microscopy, Transfection, Plasmid Preparation, Western Blot, Injection

    (A and B) Rac1 activity in control or S1PR1-depleted HPAECs following stimulation with 50 ng VEGF by using glutathione S-transferase (GST-tagged) PAK-PBD fusion protein and anti-Rac1 monoclonal antibody. Total cell lysates were immunoblotted with Rac1 or S1PR1 antibody. Representative immunoblot in (A), and (B) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 compared with siScr-transfected cells post 0, 10, and 30 min VEGF stimulation. *p < 0.05 compared with siScr post 0 or 10 min VEGF stimulation or siS1PR1-transfected cells post 0, 10, and 30 min VEGF stimulation. ###p < 0.001 and ##p < 0.01 relative to siScr-transfected cells post 0, 10, or 120 min VEGF stimulation. $$ p < 0.01 relative to siS1PR1-transfected cells post 60–120 min VEGF stimulation. (C and D) Rac1 activity in indicted ECs after treatment without or with VEGFR2 inhibitor SU5416 (100 μM) for 2 h. (C) shows the representative blot of active Rac1, and (D) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared with siScr 0 min VEGF stimulation of control treated group. ##p < 0.01 compared with siScr 60 min VEGF stimulation of control treated group. UD, undetectable. (E and F) Rac1 activity in indicated TAECs determined as described in (A). Total cell lysates were probed with S1PR1, VEGF2, and Rac1 antibodies. A representative blot is shown in (E), and (F) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs. (G) Migration of TAECs post treatment with indicated concentrations of NSC23766, a specific Rac1 inhibitor, determined as described in . One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with S1pr1 +/+ TAECs following treatment with 0, 0.1, or 1.0 μM Rac1 inhibitor; NS, compared with S1pr −/− TAECs following treatment with 0, 0.1, 1.0, or 10 μM Rac1 inhibitor. Data are representative of multiple experiments expressed as mean ± SEM. (H and I) HPAECs transfected with control (siScr) or Tiam1 siRNA for 48 h were stimulated with 50 ng VEGF, and Rac1 activity was determined as in (A). A representative blot is shown in (H), and (I) shows densitometry. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to unstimulated siScr; ##p < 0.01 relative to un-stimulated siScr transfected ECs; $$ p < 0.01 relative to siTiam1-transfected cells post 10 and 60 min VEGF stimulation. All immunoblots are representative of data from three individual experiments expressed as mean ± SEM.
    Figure Legend Snippet: (A and B) Rac1 activity in control or S1PR1-depleted HPAECs following stimulation with 50 ng VEGF by using glutathione S-transferase (GST-tagged) PAK-PBD fusion protein and anti-Rac1 monoclonal antibody. Total cell lysates were immunoblotted with Rac1 or S1PR1 antibody. Representative immunoblot in (A), and (B) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 compared with siScr-transfected cells post 0, 10, and 30 min VEGF stimulation. *p < 0.05 compared with siScr post 0 or 10 min VEGF stimulation or siS1PR1-transfected cells post 0, 10, and 30 min VEGF stimulation. ###p < 0.001 and ##p < 0.01 relative to siScr-transfected cells post 0, 10, or 120 min VEGF stimulation. $$ p < 0.01 relative to siS1PR1-transfected cells post 60–120 min VEGF stimulation. (C and D) Rac1 activity in indicted ECs after treatment without or with VEGFR2 inhibitor SU5416 (100 μM) for 2 h. (C) shows the representative blot of active Rac1, and (D) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared with siScr 0 min VEGF stimulation of control treated group. ##p < 0.01 compared with siScr 60 min VEGF stimulation of control treated group. UD, undetectable. (E and F) Rac1 activity in indicated TAECs determined as described in (A). Total cell lysates were probed with S1PR1, VEGF2, and Rac1 antibodies. A representative blot is shown in (E), and (F) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs. (G) Migration of TAECs post treatment with indicated concentrations of NSC23766, a specific Rac1 inhibitor, determined as described in . One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with S1pr1 +/+ TAECs following treatment with 0, 0.1, or 1.0 μM Rac1 inhibitor; NS, compared with S1pr −/− TAECs following treatment with 0, 0.1, 1.0, or 10 μM Rac1 inhibitor. Data are representative of multiple experiments expressed as mean ± SEM. (H and I) HPAECs transfected with control (siScr) or Tiam1 siRNA for 48 h were stimulated with 50 ng VEGF, and Rac1 activity was determined as in (A). A representative blot is shown in (H), and (I) shows densitometry. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to unstimulated siScr; ##p < 0.01 relative to un-stimulated siScr transfected ECs; $$ p < 0.01 relative to siTiam1-transfected cells post 10 and 60 min VEGF stimulation. All immunoblots are representative of data from three individual experiments expressed as mean ± SEM.

    Techniques Used: Activity Assay, Western Blot, Two Tailed Test, Transfection, Migration

    (A and B) VEGFR2 phosphorylation at Y951 or Y1175 in S1pr1 +/+ or S1pr1 −/− TAECs determined using site-specific phosphoVEGFR2 antibodies and total VEGFR2 and S1PR1 antibodies. A representative immunoblot is shown in (A), and (B) shows densitometry. Paired t test, two-tailed, ***p < 0.001 and *p < 0.05 relative to S1pr1 +/+ TAECs. (C–E) Phosphorylation of VEGFR2 in control or S1PR1-depleted HPAECs determined as described in (A) and (B). (C) shows the representative immunoblot of VEGFR2 phosphorylation at Y1175 and Y951 and total VEGFR2, and (D) and (E) show the indicated densitometries. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0 min VEGF stimulation (D). Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0, 10, 20, 60, and 120 min VEGF stimulation (E). (F and G) Y951-VEGFR2 phosphorylation in control or S1PR1-depleted HPAECs after 2 h treatment without or with SU5416. (F) shows the representative immunoblot, and (G) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to siScr control 0 min VEGF stimulated. ##p < 0.01 compared to control siS1PR1 0 and 60 min VEGF stimulation. (H and I) S1pr1 +/+ or S1pr1 −/− TAECs were treated with control anti-mouse IgG or anti-mouse-VEGF-A antibody (0.5 mg/ml) for 2h, after which VEGFR2 phosphorylation at Y951 or Y1175 and total VEGFR2 expression were determined. (H) shows a representative immunoblot, and (I) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, *** and ###p < 0.001 relative to S1pr1 +/+ TAECs following VEGF-A antibody treatment. (J) HPAECs transfected with vector, VEGFR2 cDNA, or phosphor-defective VEGFR2 cDNA (Y951F-VEGFR2 mutant) for 24 h were seeded on the polycarbonate membrane, and EC migration was determined as in . One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 relative to un-stimulated vector or VEGFR2 or Y951F-VEGFR2 cDNA transfected cells. ###p < 0.001 relative to stimulated Y951F-VEGFR2-mutant-transfected cells, **p < 0.01 relative to unstimulated VEGFR2 or Y951-VEGFR2 transfected cells. Data representative of mean ± SEM from three independent experiments. (K–N) Phosphorylation of VEGFR2 at Y1175/Y951 and Rac1 activity in HPAECs transfected with indicated mutants. (K) shows the representative immunoblotting images, and (L)–(N) show the indicated densitometries. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2-transfected cells (L). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 transfected cells. ##p < 0.01 compared to 0, 10, and 60 min VEGF-stimulated Y951F VEGFR2-transfected cells (M). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 or Y951F VEGFR2 transfected cells (N). All immunoblots represent data from three individual experiments expressed as mean ± SEM.
    Figure Legend Snippet: (A and B) VEGFR2 phosphorylation at Y951 or Y1175 in S1pr1 +/+ or S1pr1 −/− TAECs determined using site-specific phosphoVEGFR2 antibodies and total VEGFR2 and S1PR1 antibodies. A representative immunoblot is shown in (A), and (B) shows densitometry. Paired t test, two-tailed, ***p < 0.001 and *p < 0.05 relative to S1pr1 +/+ TAECs. (C–E) Phosphorylation of VEGFR2 in control or S1PR1-depleted HPAECs determined as described in (A) and (B). (C) shows the representative immunoblot of VEGFR2 phosphorylation at Y1175 and Y951 and total VEGFR2, and (D) and (E) show the indicated densitometries. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0 min VEGF stimulation (D). Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0, 10, 20, 60, and 120 min VEGF stimulation (E). (F and G) Y951-VEGFR2 phosphorylation in control or S1PR1-depleted HPAECs after 2 h treatment without or with SU5416. (F) shows the representative immunoblot, and (G) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to siScr control 0 min VEGF stimulated. ##p < 0.01 compared to control siS1PR1 0 and 60 min VEGF stimulation. (H and I) S1pr1 +/+ or S1pr1 −/− TAECs were treated with control anti-mouse IgG or anti-mouse-VEGF-A antibody (0.5 mg/ml) for 2h, after which VEGFR2 phosphorylation at Y951 or Y1175 and total VEGFR2 expression were determined. (H) shows a representative immunoblot, and (I) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, *** and ###p < 0.001 relative to S1pr1 +/+ TAECs following VEGF-A antibody treatment. (J) HPAECs transfected with vector, VEGFR2 cDNA, or phosphor-defective VEGFR2 cDNA (Y951F-VEGFR2 mutant) for 24 h were seeded on the polycarbonate membrane, and EC migration was determined as in . One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 relative to un-stimulated vector or VEGFR2 or Y951F-VEGFR2 cDNA transfected cells. ###p < 0.001 relative to stimulated Y951F-VEGFR2-mutant-transfected cells, **p < 0.01 relative to unstimulated VEGFR2 or Y951-VEGFR2 transfected cells. Data representative of mean ± SEM from three independent experiments. (K–N) Phosphorylation of VEGFR2 at Y1175/Y951 and Rac1 activity in HPAECs transfected with indicated mutants. (K) shows the representative immunoblotting images, and (L)–(N) show the indicated densitometries. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2-transfected cells (L). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 transfected cells. ##p < 0.01 compared to 0, 10, and 60 min VEGF-stimulated Y951F VEGFR2-transfected cells (M). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 or Y951F VEGFR2 transfected cells (N). All immunoblots represent data from three individual experiments expressed as mean ± SEM.

    Techniques Used: Western Blot, Two Tailed Test, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Migration, Activity Assay

    (A–D) HPAECs treated without or with pertussis toxin (PTX; 50 μM) were stimulated with VEGF for indicated time points. Phosphorylation of VEGFR2 and c-Abl1 was determined using phosphospecific antibodies. Immunoblot with c-Abl1 and VEGFR2 expression was used as loading control. (A) shows a representative immunoblot, and (B)–(D) show densitometry. One-way ANOVA, p < 0.05 (B). One-way ANOVA, p < 0.001 (C and D). Paired t test, two-tailed, ***p < 0.001 relative to unstimulated HPAECs (B–D). (E and F) Lysate from S1pr1 +/+ or S1pr1 −/− TAECs were immunoprecipitated with anti-VEGFR2 antibody, and immunocomplexes were probed with c-Abl1 or VEGFR2 antibodies. A representative immunoblot is shown in (E), and (F) shows fold change in VEGFR2-c-Abl interaction normalized against total VEGFR2. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (G and H) Lysates from indicated TAECs were immunoblotted with either phospho-Src, phospho-c-Abl1, c-Src, or c-Abl1 antibodies. A representative immunoblot is shown in (G), and (H) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (I) Indicated TAECs seeded on polycarbonate membrane were treated with or without imatinib (40 μM for 1 h), and EC migration was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 relative to untreated S1PR1 + tumor ECs; NS relative to imatinib-treated S1PR1 + or S1PR1 − TAECs. Data representative of mean ± SEM from multiple experiments. (J–M) HPAECs transfected with c-Abl1 siRNA for 72 h were stimulated with VEGF for indicated time points. VEGFR2 phosphorylation and Rac1 activity were determined as described in . Immunoblotting with c-Abl antibody was used to determine c-Abl1 depletion. Total Rac1, total VEGFR2, and actin were used as loading control. A representative immunoblot is shown in (J), and (K)–(M) show densitometries. One-way ANOVA, p < 0.01 (K–M). Paired t test, two-tailed, **p < 0.001 relative to un-stimulated siScr or c-Abl-siRNA-transfected cells; ##p < 0.01 relative to siScr HPAECs following 10 and 120 min VEGF stimulation (K–M). All western blots show data from multiple experiments expressed as mean ± SEM. (N) Model showing influence of S1P-S1PR1 signaling on VEGF-VEGFR2-mediated angiogenesis that augments EC migration and tumor vascularization and growth. Cancer cells generate S1P and VEGF. S1P ligates S1PR1 in S1PR1+ ECs. Additionally, activation of SPHK induce S1PR1 activity in a paracrine manner. S1PR1, in turn, stimulates Gi, which promotes c-Abl1 activity by VEGF. Activated c-Abl1 phosphorylates VEGFR2 on Y951, reducing VEGFR2 binding with unknown endocytic protein, causing VEGFR2 to remain on the EC surface and prolonging Rac1 activity in a Tiam1-dependent manner that increases EC migration and efficient tumor vascularization. In ECs lacking S1PR1, VEGF leads to VEGFR2 phosphorylation at Y1175, followed by receptor internalization. Internalized receptor induces ERK activity but transiently activates Rac1, leading to reduced EC migration and impairing tumor vascularization and growth.
    Figure Legend Snippet: (A–D) HPAECs treated without or with pertussis toxin (PTX; 50 μM) were stimulated with VEGF for indicated time points. Phosphorylation of VEGFR2 and c-Abl1 was determined using phosphospecific antibodies. Immunoblot with c-Abl1 and VEGFR2 expression was used as loading control. (A) shows a representative immunoblot, and (B)–(D) show densitometry. One-way ANOVA, p < 0.05 (B). One-way ANOVA, p < 0.001 (C and D). Paired t test, two-tailed, ***p < 0.001 relative to unstimulated HPAECs (B–D). (E and F) Lysate from S1pr1 +/+ or S1pr1 −/− TAECs were immunoprecipitated with anti-VEGFR2 antibody, and immunocomplexes were probed with c-Abl1 or VEGFR2 antibodies. A representative immunoblot is shown in (E), and (F) shows fold change in VEGFR2-c-Abl interaction normalized against total VEGFR2. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (G and H) Lysates from indicated TAECs were immunoblotted with either phospho-Src, phospho-c-Abl1, c-Src, or c-Abl1 antibodies. A representative immunoblot is shown in (G), and (H) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (I) Indicated TAECs seeded on polycarbonate membrane were treated with or without imatinib (40 μM for 1 h), and EC migration was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 relative to untreated S1PR1 + tumor ECs; NS relative to imatinib-treated S1PR1 + or S1PR1 − TAECs. Data representative of mean ± SEM from multiple experiments. (J–M) HPAECs transfected with c-Abl1 siRNA for 72 h were stimulated with VEGF for indicated time points. VEGFR2 phosphorylation and Rac1 activity were determined as described in . Immunoblotting with c-Abl antibody was used to determine c-Abl1 depletion. Total Rac1, total VEGFR2, and actin were used as loading control. A representative immunoblot is shown in (J), and (K)–(M) show densitometries. One-way ANOVA, p < 0.01 (K–M). Paired t test, two-tailed, **p < 0.001 relative to un-stimulated siScr or c-Abl-siRNA-transfected cells; ##p < 0.01 relative to siScr HPAECs following 10 and 120 min VEGF stimulation (K–M). All western blots show data from multiple experiments expressed as mean ± SEM. (N) Model showing influence of S1P-S1PR1 signaling on VEGF-VEGFR2-mediated angiogenesis that augments EC migration and tumor vascularization and growth. Cancer cells generate S1P and VEGF. S1P ligates S1PR1 in S1PR1+ ECs. Additionally, activation of SPHK induce S1PR1 activity in a paracrine manner. S1PR1, in turn, stimulates Gi, which promotes c-Abl1 activity by VEGF. Activated c-Abl1 phosphorylates VEGFR2 on Y951, reducing VEGFR2 binding with unknown endocytic protein, causing VEGFR2 to remain on the EC surface and prolonging Rac1 activity in a Tiam1-dependent manner that increases EC migration and efficient tumor vascularization. In ECs lacking S1PR1, VEGF leads to VEGFR2 phosphorylation at Y1175, followed by receptor internalization. Internalized receptor induces ERK activity but transiently activates Rac1, leading to reduced EC migration and impairing tumor vascularization and growth.

    Techniques Used: Western Blot, Expressing, Two Tailed Test, Immunoprecipitation, Migration, Transfection, Activity Assay, Activation Assay, Binding Assay

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Labeling, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Software

    anti sphingosine 1 phosphate receptor 1 antibody  (Alomone Labs)


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    Alomone Labs anti sphingosine 1 phosphate receptor 1 antibody
    Representative blots of <t>S1PR1</t> levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG35–55–treated animals that received vehicle.
    Anti Sphingosine 1 Phosphate Receptor 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti sphingosine 1 phosphate receptor 1 antibody - by Bioz Stars, 2023-01
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    Images

    1) Product Images from "Fingolimod Reduces Neuropathic Pain Behaviors in a Mouse Model of Multiple Sclerosis by a Sphingosine-1 Phosphate Receptor 1-Dependent Inhibition of Central Sensitization in the Dorsal Horn."

    Article Title: Fingolimod Reduces Neuropathic Pain Behaviors in a Mouse Model of Multiple Sclerosis by a Sphingosine-1 Phosphate Receptor 1-Dependent Inhibition of Central Sensitization in the Dorsal Horn.

    Journal: Pain

    doi: 10.1097/j.pain.0000000000001106

    Representative blots of S1PR1 levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG35–55–treated animals that received vehicle.
    Figure Legend Snippet: Representative blots of S1PR1 levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG35–55–treated animals that received vehicle.

    Techniques Used: Expressing

    anti s1p1  (Alomone Labs)


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    Alomone Labs anti s1p1
    AD2900 shows antagonistic activities against <t> S1P1, </t> 2, 3, 4, and 5
    Anti S1p1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes"

    Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18626

    AD2900 shows antagonistic activities against  S1P1,  2, 3, 4, and 5
    Figure Legend Snippet: AD2900 shows antagonistic activities against S1P1, 2, 3, 4, and 5

    Techniques Used:

    (A, B) The percentages of S1P1-positive PBMCs after the treatment with different concentrations of AD2900, FTY720, or SEW2871 and at different time points were examined by FACS analysis. S1P1 expression was tested in PBMCs after a 30-min treatment with AD2900 at different concentrations (A) or after a 30-min or 60-min treatment with 100 nM AD2900, FTY720, or SEW2871 (B) . (C) The percentage of CCR7-positive PBMCs was tested by FACS analysis after a 30-min treatment with 100 nM AD2900, FTY720, or SEW2871. All the significances are compared to untreated PBMCs. Results summarize the results of at least four independent experiments. Results of Student’s t -test: *(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).
    Figure Legend Snippet: (A, B) The percentages of S1P1-positive PBMCs after the treatment with different concentrations of AD2900, FTY720, or SEW2871 and at different time points were examined by FACS analysis. S1P1 expression was tested in PBMCs after a 30-min treatment with AD2900 at different concentrations (A) or after a 30-min or 60-min treatment with 100 nM AD2900, FTY720, or SEW2871 (B) . (C) The percentage of CCR7-positive PBMCs was tested by FACS analysis after a 30-min treatment with 100 nM AD2900, FTY720, or SEW2871. All the significances are compared to untreated PBMCs. Results summarize the results of at least four independent experiments. Results of Student’s t -test: *(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

    Techniques Used: Expressing, Two Tailed Test

    C57BL/6 mice were orally administered with 1.8, 2.7, and 3.6 mg/l AD2900 or 1.8 mg/l FTY720 for 2 days, as shown in Figure . Leukocytes from blood, spleen, and pLNs were collected and stained with CD3e and S1P1 or CCR7 fluorescent antibodies and then analyzed by FACS analysis. The percentages of S1P1+ CD3e+ T cells from blood (A) , spleen (B) , and pLNs (C) are shown. The percentages of CCR7+ CD3e+ T cells from blood (D) , spleen (E) , and pLNs (F) are shown. All the significances are compared to untreated healthy mice. Results summarize at least three independent experiments. Results of Student’s t -test:*(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).
    Figure Legend Snippet: C57BL/6 mice were orally administered with 1.8, 2.7, and 3.6 mg/l AD2900 or 1.8 mg/l FTY720 for 2 days, as shown in Figure . Leukocytes from blood, spleen, and pLNs were collected and stained with CD3e and S1P1 or CCR7 fluorescent antibodies and then analyzed by FACS analysis. The percentages of S1P1+ CD3e+ T cells from blood (A) , spleen (B) , and pLNs (C) are shown. The percentages of CCR7+ CD3e+ T cells from blood (D) , spleen (E) , and pLNs (F) are shown. All the significances are compared to untreated healthy mice. Results summarize at least three independent experiments. Results of Student’s t -test:*(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

    Techniques Used: Staining, Two Tailed Test

    As an antagonist to S1P receptors 1–5, AD2900 can compete with S1P to bind S1P receptors leading to reduced S1P signaling and enhanced expression of S1P1 on T cells in S1P-rich environments such as the blood and the spleen. This altered expression, together with decreased CCR7 expression, inhibits T-cell entry into the lymph nodes (LNs) from the blood, causing accumulation of T cells in the blood. However, the entry of T cells to the spleen is not affected because it is not S1P dependent. Since Tcm-like cells express CCR7, these cells are attracted to the spleen and accumulate in it; yet, S1P1 elevated expression may have an effect on the S1P-dependent ingression of these cells from the MZ to the white pulp. Tef/em-like cells, which are CCR7 negative, are the primary T-cell subpopulation in the blood after AD2900 treatment. The significant decrease in naive T-cell counts in the circulation and peripheral lymphoid tissues tested may be explained by the inhibition of S1P signaling in the thymus leading to attenuated T-cell egression from the thymus to the circulation. Arrow key: thick = response; dashed = inhibition.
    Figure Legend Snippet: As an antagonist to S1P receptors 1–5, AD2900 can compete with S1P to bind S1P receptors leading to reduced S1P signaling and enhanced expression of S1P1 on T cells in S1P-rich environments such as the blood and the spleen. This altered expression, together with decreased CCR7 expression, inhibits T-cell entry into the lymph nodes (LNs) from the blood, causing accumulation of T cells in the blood. However, the entry of T cells to the spleen is not affected because it is not S1P dependent. Since Tcm-like cells express CCR7, these cells are attracted to the spleen and accumulate in it; yet, S1P1 elevated expression may have an effect on the S1P-dependent ingression of these cells from the MZ to the white pulp. Tef/em-like cells, which are CCR7 negative, are the primary T-cell subpopulation in the blood after AD2900 treatment. The significant decrease in naive T-cell counts in the circulation and peripheral lymphoid tissues tested may be explained by the inhibition of S1P signaling in the thymus leading to attenuated T-cell egression from the thymus to the circulation. Arrow key: thick = response; dashed = inhibition.

    Techniques Used: Expressing, Inhibition

    rabbit anti s1pr1  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti s1pr1
    Rabbit Anti S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    Alomone Labs rabbit anti s1pr1
    Hypothetical mechanism by which CNTF/Stat3 and <t>S1P/S1PR1</t> interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.
    Rabbit Anti S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti s1pr1 antibody
    Immunohistochemistry (IHC) of <t>S1PR1</t> in postmortem DLPFC tissues from the representative normal control and schizophrenia Type 1 and Type 2.
    Anti S1pr1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs s1pr1
    OCTA images under influence of pro-and <t>anti-S1PR1</t> agent. (Note that all OCTA images shown above may contain artifact from OCTA itself). A: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the no PC model. Upper row: sham model (no PC and no drug administration). Middle row: sham + SEW model (no PC and SEW administration). Lower row: sham + VPC model (no PC and VPC administration). The appearance of the retinal blood vessels did not change, and no collateral vessel formation occurred in the sham model, sham + SEW administration, and sham + VPC administration groups (images show the deep capillary bed of the retina). B: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the RVO model. Upper row: normal RVO model (no drug administration). Middle row: RVO + SEW (SEW-administration). Lower row: RVO + VPC (VPC-administration). The number of collateral vessels in the RVO + SEW group was significantly greater than in the normal RVO group, and RVO + VPC group (both, p < 0.0001). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although this difference was not statistically significant (p = 0.1427). Right schema: the red cross shows the occlusion point. Black radial lines represent the retinal vessels. The blue tortured line represents the newly formed collateral vessels. As illustrated in the right schema, the RVO + SEW group tended to have more collateral vessels than the normal RVO group, and the RVO + VPC group tended to have fewer collateral vessels than the normal RVO group.
    S1pr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti sphingosine 1 phosphate receptor 1 extracellular
    (A and B) After 24 h post transfection of HPAECs with either scrambled siRNA (siScr) or <t>S1PR1</t> siRNA (siS1PR1), these cells were transduced with control vector or hemagglutinin (HA)-tagged S1PR1 cDNA to restore S1PR1 expression. Cells were stimulated with VEGF (50 ng) with or without S1P (1 μM), and network formation was assessed at 16 h by light microscopy (20× and 40×). (A) shows a representative image taken at 40× magnification, whereas (B) shows quantitation of the endothelial polygonal areas formed using 20× magnification. Bars, 50 μm (A). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to un-stimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (C) HPAECs processed as described in (B) were seeded on the top surface of Matrigel-coated polycarbonate membrane inserts of Boyden chamber. Cells were incubated with basal media along with indicated ligands (50 ng VEGF or 1 μM S1P) placed outside the chambers. After 16 h, ECs migrating to the bottom surface of the insert were fixed and nuclei were stained with DAPI, and their migration was determined using confocal microscope (20×). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to unstimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (D) Tumor growth in S1pr1 fl/fl versus EC-S1pr1 −/− mice following subcutaneous injection of 0.25 × 10 6 Lewis lung cell carcinoma (LLC) cells. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to S1pr1 fl/fl mice. Data expressed as mean ± SEM. (E) Tumor weight post day 21 of LLC cell injection in indicated strains of mice. Inset: a representative image of tumor from each strain. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 compared to S1pr1 fl/fl tumors. (F–H) S1pr1 fl/fl and EC-S1pr1 −/− tumor-associated ECs (TAECs) were isolated using FACS and cultured, and network formation and migration were assessed as described in (A)–(C). (F) shows the representative images of network formation; (G) shows the quantification of number of branch points formed; and (H) shows the quantification of the migrated cells per field. Bars, 50 μm. Image representative of multiple samples from different mice (F). Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs (G and H). All data represented were repeated multiple times and are expressed as mean ± SEM.
    Anti Sphingosine 1 Phosphate Receptor 1 Extracellular, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti sphingosine 1 phosphate receptor 1 antibody
    Representative blots of <t>S1PR1</t> levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG35–55–treated animals that received vehicle.
    Anti Sphingosine 1 Phosphate Receptor 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Alomone Labs anti s1p1
    AD2900 shows antagonistic activities against <t> S1P1, </t> 2, 3, 4, and 5
    Anti S1p1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hypothetical mechanism by which CNTF/Stat3 and S1P/S1PR1 interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.

    Journal: Neural Plasticity

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    doi: 10.1155/2017/6818970

    Figure Lengend Snippet: Hypothetical mechanism by which CNTF/Stat3 and S1P/S1PR1 interaction may orchestrate neuronal survival and axonal growth. CNTF binds and activates a heterotrimeric receptor complex, composed of CNTFR α , leukemia inhibitory factor receptor (LIFR), and gp130, leading to Stat3 phosphorylation (P-Stat3) and activation. (a) P-Stat3-driven transcription may increase the expression of S1PR1 and its translocation to the plasma membrane. The activation of S1PR1 by S1P may trigger downstream growth mechanisms resulting in (b) neuronal survival and (c) axonal growth.

    Article Snippet: Primary antibodies were rabbit anti- β 3-tubulin (1 : 1000; ab18207, Abcam), mouse anti- β 3-tubulin (1 : 1000; G712A, Promega, Madison, WI, USA), rabbit anti-S1PR1 (1 : 50; ASR-011; Alomone Labs, Jerusalem, Israel), rabbit anti-S1PR1 (1 : 200; PA1-1040, Life Technologies), and rabbit anti-phospho-Stat3 (1 : 100; 9131, Cell Signaling, Whitby, ON, Canada).

    Techniques: Activation Assay, Expressing, Translocation Assay

    S1PR1 knockdown potentiates CNTF-induced axonal regeneration. (a) Axonal regeneration was visualized on longitudinal sections of optic nerves two weeks after crush injury and 4 weeks after coinfection with ShH10.CNTF and AAV2 vectors. Axons were traced with cholera toxin β subunit (CTb) conjugated to Alexa 594 the day before tissue fixation. (b) The infection of retinal cells with ShH10.CNTF and AAV2.shRNA-S1PR1 promoted lengthy axonal regeneration in the optic nerve compared with the ShH10.CNTF/AAV2.GFP combination. (c) Quantitatively, axonal fibers were significantly more numerous between 1300 and 1800 μ m past the lesion site with ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 6 mice) than with ShH10.CNTF/AAV2.GFP ( n = 5 mice) treatments (ANOVA, ∗ p < 0.05). ShH10.CNTF/AAV2.S1PR1 did not influence axonal regeneration ( n = 6 mice). (d) The measurement of the longest axons revealed better growth distances in ShH10.CNTF-/AAV2.shRNA-S1PR1-treated animals than in mice receiving ShH10.CNTF/AAV2.GFP. Scale bars: (b) top = 200 μ m; (b) bottom = 100 μ m.

    Journal: Neural Plasticity

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    doi: 10.1155/2017/6818970

    Figure Lengend Snippet: S1PR1 knockdown potentiates CNTF-induced axonal regeneration. (a) Axonal regeneration was visualized on longitudinal sections of optic nerves two weeks after crush injury and 4 weeks after coinfection with ShH10.CNTF and AAV2 vectors. Axons were traced with cholera toxin β subunit (CTb) conjugated to Alexa 594 the day before tissue fixation. (b) The infection of retinal cells with ShH10.CNTF and AAV2.shRNA-S1PR1 promoted lengthy axonal regeneration in the optic nerve compared with the ShH10.CNTF/AAV2.GFP combination. (c) Quantitatively, axonal fibers were significantly more numerous between 1300 and 1800 μ m past the lesion site with ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 6 mice) than with ShH10.CNTF/AAV2.GFP ( n = 5 mice) treatments (ANOVA, ∗ p < 0.05). ShH10.CNTF/AAV2.S1PR1 did not influence axonal regeneration ( n = 6 mice). (d) The measurement of the longest axons revealed better growth distances in ShH10.CNTF-/AAV2.shRNA-S1PR1-treated animals than in mice receiving ShH10.CNTF/AAV2.GFP. Scale bars: (b) top = 200 μ m; (b) bottom = 100 μ m.

    Article Snippet: Primary antibodies were rabbit anti- β 3-tubulin (1 : 1000; ab18207, Abcam), mouse anti- β 3-tubulin (1 : 1000; G712A, Promega, Madison, WI, USA), rabbit anti-S1PR1 (1 : 50; ASR-011; Alomone Labs, Jerusalem, Israel), rabbit anti-S1PR1 (1 : 200; PA1-1040, Life Technologies), and rabbit anti-phospho-Stat3 (1 : 100; 9131, Cell Signaling, Whitby, ON, Canada).

    Techniques: Infection, shRNA

    Modulation of S1PR1 expression by the CNTF/Stat3 pathway in optic nerve-injured retinae. (a, b) S1PR1 expression changes were monitored 3 d after ONC in retinae infected with ShH10.CNTF or ShH10.Empty, a control vector that was deprived of cDNA sequence. ShH10 viruses preferentially infected Müller glia in the retina . P-Stat3 and S1PR1 were markedly increased by ShH10.CNTF in RGC somata identified using β 3-tubulin as a specific marker. (c) Five days after ONC, qRT-PCR measurements showed that the infection of RGCs with AAV2.Stat3 significantly increased the mRNA level of S1pr1 compared with control AAV2.GFP vector. ShH10 and AAV2 viruses were intravitreally injected 4 weeks before ONC. Three mice were analyzed/grouped. Statistics: one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; NS: not significant. Scale bar: 50 μ m.

    Journal: Neural Plasticity

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    doi: 10.1155/2017/6818970

    Figure Lengend Snippet: Modulation of S1PR1 expression by the CNTF/Stat3 pathway in optic nerve-injured retinae. (a, b) S1PR1 expression changes were monitored 3 d after ONC in retinae infected with ShH10.CNTF or ShH10.Empty, a control vector that was deprived of cDNA sequence. ShH10 viruses preferentially infected Müller glia in the retina . P-Stat3 and S1PR1 were markedly increased by ShH10.CNTF in RGC somata identified using β 3-tubulin as a specific marker. (c) Five days after ONC, qRT-PCR measurements showed that the infection of RGCs with AAV2.Stat3 significantly increased the mRNA level of S1pr1 compared with control AAV2.GFP vector. ShH10 and AAV2 viruses were intravitreally injected 4 weeks before ONC. Three mice were analyzed/grouped. Statistics: one-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; NS: not significant. Scale bar: 50 μ m.

    Article Snippet: Primary antibodies were rabbit anti- β 3-tubulin (1 : 1000; ab18207, Abcam), mouse anti- β 3-tubulin (1 : 1000; G712A, Promega, Madison, WI, USA), rabbit anti-S1PR1 (1 : 50; ASR-011; Alomone Labs, Jerusalem, Israel), rabbit anti-S1PR1 (1 : 200; PA1-1040, Life Technologies), and rabbit anti-phospho-Stat3 (1 : 100; 9131, Cell Signaling, Whitby, ON, Canada).

    Techniques: Expressing, Infection, Plasmid Preparation, Sequencing, Marker, Quantitative RT-PCR, Injection

    S1PR1 knockdown alters CNTF-induced RGC survival after ONC. (a) Two weeks after ONC, surviving RGCs were observed in retinal flat-mounts after immunofluorescent staining for β 3-tubulin. Less RGCs were visible in retinae infected with AAV2.shRNA-S1PR1 and ShH10.CNTF ( n = 5 mice) than in mice injected with ShH10.CNTF/AAV2.GFP ( n = 7 mice) or ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 5 mice). (b) Quantitatively, the average number of surviving RGCs was statistically lower in whole retinae transduced with ShH10.CNTF/AAV2.shRNA-S1PR1 than in the two other groups of animals (ANOVA, ∗∗∗ p < 0.001). (c) The reduction of RGC survival caused by ShH10.CNTF/AAV2.shRNA-S1PR1 was the most pronounced in the superior quadrant of the retina (ANOVA, ∗∗∗ p < 0.001). Scale bar: 100 μ m.

    Journal: Neural Plasticity

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    doi: 10.1155/2017/6818970

    Figure Lengend Snippet: S1PR1 knockdown alters CNTF-induced RGC survival after ONC. (a) Two weeks after ONC, surviving RGCs were observed in retinal flat-mounts after immunofluorescent staining for β 3-tubulin. Less RGCs were visible in retinae infected with AAV2.shRNA-S1PR1 and ShH10.CNTF ( n = 5 mice) than in mice injected with ShH10.CNTF/AAV2.GFP ( n = 7 mice) or ShH10.CNTF/AAV2.shRNA-S1PR1 ( n = 5 mice). (b) Quantitatively, the average number of surviving RGCs was statistically lower in whole retinae transduced with ShH10.CNTF/AAV2.shRNA-S1PR1 than in the two other groups of animals (ANOVA, ∗∗∗ p < 0.001). (c) The reduction of RGC survival caused by ShH10.CNTF/AAV2.shRNA-S1PR1 was the most pronounced in the superior quadrant of the retina (ANOVA, ∗∗∗ p < 0.001). Scale bar: 100 μ m.

    Article Snippet: Primary antibodies were rabbit anti- β 3-tubulin (1 : 1000; ab18207, Abcam), mouse anti- β 3-tubulin (1 : 1000; G712A, Promega, Madison, WI, USA), rabbit anti-S1PR1 (1 : 50; ASR-011; Alomone Labs, Jerusalem, Israel), rabbit anti-S1PR1 (1 : 200; PA1-1040, Life Technologies), and rabbit anti-phospho-Stat3 (1 : 100; 9131, Cell Signaling, Whitby, ON, Canada).

    Techniques: Staining, Infection, shRNA, Injection, Transduction

    The expression of P-Stat3 is not changed by S1PR1 silencing after CNTF stimulation. The expression of P-Stat3 was assessed by western blotting in protein lysates (20 μ g) from retinae treated with ShH10 and AAV2 viruses. P-Stat3 and Stat3 blots were quantified by densitometry using the ImageJ software (NIH). The level of P-Stat3/Stat3 was not significantly different between mice treated with ShH10.CNTF/AAV2.shRNA-S1PR1 and ShH10.CNTF/AAV2.GFP. Three mice were analyzed for each group.

    Journal: Neural Plasticity

    Article Title: Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

    doi: 10.1155/2017/6818970

    Figure Lengend Snippet: The expression of P-Stat3 is not changed by S1PR1 silencing after CNTF stimulation. The expression of P-Stat3 was assessed by western blotting in protein lysates (20 μ g) from retinae treated with ShH10 and AAV2 viruses. P-Stat3 and Stat3 blots were quantified by densitometry using the ImageJ software (NIH). The level of P-Stat3/Stat3 was not significantly different between mice treated with ShH10.CNTF/AAV2.shRNA-S1PR1 and ShH10.CNTF/AAV2.GFP. Three mice were analyzed for each group.

    Article Snippet: Primary antibodies were rabbit anti- β 3-tubulin (1 : 1000; ab18207, Abcam), mouse anti- β 3-tubulin (1 : 1000; G712A, Promega, Madison, WI, USA), rabbit anti-S1PR1 (1 : 50; ASR-011; Alomone Labs, Jerusalem, Israel), rabbit anti-S1PR1 (1 : 200; PA1-1040, Life Technologies), and rabbit anti-phospho-Stat3 (1 : 100; 9131, Cell Signaling, Whitby, ON, Canada).

    Techniques: Expressing, Western Blot, Software, shRNA

    Immunohistochemistry (IHC) of S1PR1 in postmortem DLPFC tissues from the representative normal control and schizophrenia Type 1 and Type 2.

    Journal: Frontiers in Psychiatry

    Article Title: Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2

    doi: 10.3389/fpsyt.2022.827981

    Figure Lengend Snippet: Immunohistochemistry (IHC) of S1PR1 in postmortem DLPFC tissues from the representative normal control and schizophrenia Type 1 and Type 2.

    Article Snippet: After that, all sections were stained with anti-S1PR1 antibody (Alomone, Jerusalem, Israel) overnight at 4°C, washed and followed by incubation with ImmPRESS HRP Horse anti-rabbit polymer for 1 h at RT, and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA).

    Techniques: Immunohistochemistry

    Representative images of [ 3 H]CS1P1 autoradiograph, S1PR1 immunostaining, and Hematoxylin and eosin (H&E) staining in postmortem human DLPFC tissues. The distribution of [ 3 H]CS1P1 matched well with anti-S1PR1 antibody, and was mainly located in the gray matter regions as indicated in the H&E staining.

    Journal: Frontiers in Psychiatry

    Article Title: Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2

    doi: 10.3389/fpsyt.2022.827981

    Figure Lengend Snippet: Representative images of [ 3 H]CS1P1 autoradiograph, S1PR1 immunostaining, and Hematoxylin and eosin (H&E) staining in postmortem human DLPFC tissues. The distribution of [ 3 H]CS1P1 matched well with anti-S1PR1 antibody, and was mainly located in the gray matter regions as indicated in the H&E staining.

    Article Snippet: After that, all sections were stained with anti-S1PR1 antibody (Alomone, Jerusalem, Israel) overnight at 4°C, washed and followed by incubation with ImmPRESS HRP Horse anti-rabbit polymer for 1 h at RT, and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA).

    Techniques: Autoradiography, Immunostaining, Staining

    Autoradiography images of S1PR1 using [ 3 H]CS1P1 in postmortem DLPFC tissues from representative normal control, schizophrenia Type 1, and schizophrenia Type 2. In general, [ 3 H]CS1P1 was higher in Type 2 schizophrenia subjects compared with normal control and Type 1 schizophrenia subjects.

    Journal: Frontiers in Psychiatry

    Article Title: Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2

    doi: 10.3389/fpsyt.2022.827981

    Figure Lengend Snippet: Autoradiography images of S1PR1 using [ 3 H]CS1P1 in postmortem DLPFC tissues from representative normal control, schizophrenia Type 1, and schizophrenia Type 2. In general, [ 3 H]CS1P1 was higher in Type 2 schizophrenia subjects compared with normal control and Type 1 schizophrenia subjects.

    Article Snippet: After that, all sections were stained with anti-S1PR1 antibody (Alomone, Jerusalem, Israel) overnight at 4°C, washed and followed by incubation with ImmPRESS HRP Horse anti-rabbit polymer for 1 h at RT, and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA).

    Techniques: Autoradiography

    ARG S1PR1 intensity expression (fmol/mg) triplicate measures (M1, M2, and M3) in the DLPFC from normal controls, schizophrenia Type 1, and schizophrenia Type 2.

    Journal: Frontiers in Psychiatry

    Article Title: Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2

    doi: 10.3389/fpsyt.2022.827981

    Figure Lengend Snippet: ARG S1PR1 intensity expression (fmol/mg) triplicate measures (M1, M2, and M3) in the DLPFC from normal controls, schizophrenia Type 1, and schizophrenia Type 2.

    Article Snippet: After that, all sections were stained with anti-S1PR1 antibody (Alomone, Jerusalem, Israel) overnight at 4°C, washed and followed by incubation with ImmPRESS HRP Horse anti-rabbit polymer for 1 h at RT, and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA).

    Techniques: Expressing

    ARG S1PR1 intensity expression (in fmol/mg) comparison between normal controls, schizophrenia Type 1, and schizophrenia Type 2 (* p < 0.05).

    Journal: Frontiers in Psychiatry

    Article Title: Differential Sphingosine-1-Phosphate Receptor-1 Protein Expression in the Dorsolateral Prefrontal Cortex Between Schizophrenia Type 1 and Type 2

    doi: 10.3389/fpsyt.2022.827981

    Figure Lengend Snippet: ARG S1PR1 intensity expression (in fmol/mg) comparison between normal controls, schizophrenia Type 1, and schizophrenia Type 2 (* p < 0.05).

    Article Snippet: After that, all sections were stained with anti-S1PR1 antibody (Alomone, Jerusalem, Israel) overnight at 4°C, washed and followed by incubation with ImmPRESS HRP Horse anti-rabbit polymer for 1 h at RT, and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA).

    Techniques: Expressing

    OCTA images under influence of pro-and anti-S1PR1 agent. (Note that all OCTA images shown above may contain artifact from OCTA itself). A: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the no PC model. Upper row: sham model (no PC and no drug administration). Middle row: sham + SEW model (no PC and SEW administration). Lower row: sham + VPC model (no PC and VPC administration). The appearance of the retinal blood vessels did not change, and no collateral vessel formation occurred in the sham model, sham + SEW administration, and sham + VPC administration groups (images show the deep capillary bed of the retina). B: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the RVO model. Upper row: normal RVO model (no drug administration). Middle row: RVO + SEW (SEW-administration). Lower row: RVO + VPC (VPC-administration). The number of collateral vessels in the RVO + SEW group was significantly greater than in the normal RVO group, and RVO + VPC group (both, p < 0.0001). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although this difference was not statistically significant (p = 0.1427). Right schema: the red cross shows the occlusion point. Black radial lines represent the retinal vessels. The blue tortured line represents the newly formed collateral vessels. As illustrated in the right schema, the RVO + SEW group tended to have more collateral vessels than the normal RVO group, and the RVO + VPC group tended to have fewer collateral vessels than the normal RVO group.

    Journal: Heliyon

    Article Title: Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation

    doi: 10.1016/j.heliyon.2021.e05902

    Figure Lengend Snippet: OCTA images under influence of pro-and anti-S1PR1 agent. (Note that all OCTA images shown above may contain artifact from OCTA itself). A: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the no PC model. Upper row: sham model (no PC and no drug administration). Middle row: sham + SEW model (no PC and SEW administration). Lower row: sham + VPC model (no PC and VPC administration). The appearance of the retinal blood vessels did not change, and no collateral vessel formation occurred in the sham model, sham + SEW administration, and sham + VPC administration groups (images show the deep capillary bed of the retina). B: OCTA imaging of S1PR1 agonist and inverse agonist administration in the deep capillary bed of the retina in the RVO model. Upper row: normal RVO model (no drug administration). Middle row: RVO + SEW (SEW-administration). Lower row: RVO + VPC (VPC-administration). The number of collateral vessels in the RVO + SEW group was significantly greater than in the normal RVO group, and RVO + VPC group (both, p < 0.0001). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although this difference was not statistically significant (p = 0.1427). Right schema: the red cross shows the occlusion point. Black radial lines represent the retinal vessels. The blue tortured line represents the newly formed collateral vessels. As illustrated in the right schema, the RVO + SEW group tended to have more collateral vessels than the normal RVO group, and the RVO + VPC group tended to have fewer collateral vessels than the normal RVO group.

    Article Snippet: The following antibodies were used for wholemount immunolabeling of the retina: anti-CD31 monoclonal antibody (rat anti-mouse CD31, 1:500; BD Biosciences Pharmingen, San Diego, CA, USA) was used to detect blood vessels with Alexa Fluor-568 secondary antibody (1:400); anti-S1PR1 monoclonal antibody (rabbit anti-mouse, rat S1PR1, 1:200; Almone Labs, Jerusalem, MA, USA) was used to detect S1PR1 with Alexa Fluor-488 secondary antibody (1:400).

    Techniques: Imaging

    A: Average number of collateral vessels per eye. The average number of collateral vessels per eye was 1.66 in the normal RVO group, 4.11 in the RVO + SEW group, and 0.71 in the RVO + VPC group. The number of collateral vessels differed among the normal RVO group, RVO + SEW group, and RVO + VPC group (p < 0.0001). In addition, the number of collateral vessels in the RVO + SEW group was also significantly higher than in the normal RVO group and RVO + VPC group (p < 0.0001 for both groups). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although the difference was not statistically significant (p = 0.1427). B: RT-PCR for S1PR1 expression in the whole retina. Differences in the mean levels of S1PR1 expression were observed between the control group (no laser irradiation and no drug administration), and at time points 6 h, 12 h, 24 h, and 3 days after laser irradiation (p < 0.0001). The levels of S1PR1 expression 6 h (p < 0.0001) and 12 h (p = 0.0023) after laser irradiation were significantly higher than the sham, and these levels then gradually decreased and did not increase thereafter. When comparing S1PR1 expression levels between 6 h and 12 h post-laser irradiation, the expression at 6 h was significantly greater than at 12 h (p < 0.0001). The expression of S1PR1 mRNA peaked by about 6 h after blood vessel occlusion, and was significantly greater than control levels.

    Journal: Heliyon

    Article Title: Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation

    doi: 10.1016/j.heliyon.2021.e05902

    Figure Lengend Snippet: A: Average number of collateral vessels per eye. The average number of collateral vessels per eye was 1.66 in the normal RVO group, 4.11 in the RVO + SEW group, and 0.71 in the RVO + VPC group. The number of collateral vessels differed among the normal RVO group, RVO + SEW group, and RVO + VPC group (p < 0.0001). In addition, the number of collateral vessels in the RVO + SEW group was also significantly higher than in the normal RVO group and RVO + VPC group (p < 0.0001 for both groups). The number of collateral vessels in the RVO + VPC group tended to be smaller than in the normal RVO group, although the difference was not statistically significant (p = 0.1427). B: RT-PCR for S1PR1 expression in the whole retina. Differences in the mean levels of S1PR1 expression were observed between the control group (no laser irradiation and no drug administration), and at time points 6 h, 12 h, 24 h, and 3 days after laser irradiation (p < 0.0001). The levels of S1PR1 expression 6 h (p < 0.0001) and 12 h (p = 0.0023) after laser irradiation were significantly higher than the sham, and these levels then gradually decreased and did not increase thereafter. When comparing S1PR1 expression levels between 6 h and 12 h post-laser irradiation, the expression at 6 h was significantly greater than at 12 h (p < 0.0001). The expression of S1PR1 mRNA peaked by about 6 h after blood vessel occlusion, and was significantly greater than control levels.

    Article Snippet: The following antibodies were used for wholemount immunolabeling of the retina: anti-CD31 monoclonal antibody (rat anti-mouse CD31, 1:500; BD Biosciences Pharmingen, San Diego, CA, USA) was used to detect blood vessels with Alexa Fluor-568 secondary antibody (1:400); anti-S1PR1 monoclonal antibody (rabbit anti-mouse, rat S1PR1, 1:200; Almone Labs, Jerusalem, MA, USA) was used to detect S1PR1 with Alexa Fluor-488 secondary antibody (1:400).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Irradiation

    Immunofluorescence imaging of retinal flat mounts. a: Immunohistochemical staining for CD31 at day 1 post-PC. b: Immunohistochemical staining for S1PR1 at day 1 post-PC. c: Immunohistochemical staining for CD31 at day 3 post-PC. d: Immunohistochemical staining for S1PR1 at day 3 post-PC. White oval: occluded vein. On day 1 post-PC, S1PR1 staining occurred along the laser-occluded blood vessel, but was not present along other blood vessels. There was no prominent staining for S1PR1 along the occluded blood vessels after the first day post-PC.

    Journal: Heliyon

    Article Title: Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation

    doi: 10.1016/j.heliyon.2021.e05902

    Figure Lengend Snippet: Immunofluorescence imaging of retinal flat mounts. a: Immunohistochemical staining for CD31 at day 1 post-PC. b: Immunohistochemical staining for S1PR1 at day 1 post-PC. c: Immunohistochemical staining for CD31 at day 3 post-PC. d: Immunohistochemical staining for S1PR1 at day 3 post-PC. White oval: occluded vein. On day 1 post-PC, S1PR1 staining occurred along the laser-occluded blood vessel, but was not present along other blood vessels. There was no prominent staining for S1PR1 along the occluded blood vessels after the first day post-PC.

    Article Snippet: The following antibodies were used for wholemount immunolabeling of the retina: anti-CD31 monoclonal antibody (rat anti-mouse CD31, 1:500; BD Biosciences Pharmingen, San Diego, CA, USA) was used to detect blood vessels with Alexa Fluor-568 secondary antibody (1:400); anti-S1PR1 monoclonal antibody (rabbit anti-mouse, rat S1PR1, 1:200; Almone Labs, Jerusalem, MA, USA) was used to detect S1PR1 with Alexa Fluor-488 secondary antibody (1:400).

    Techniques: Immunofluorescence, Imaging, Immunohistochemical staining, Staining

    (A and B) After 24 h post transfection of HPAECs with either scrambled siRNA (siScr) or S1PR1 siRNA (siS1PR1), these cells were transduced with control vector or hemagglutinin (HA)-tagged S1PR1 cDNA to restore S1PR1 expression. Cells were stimulated with VEGF (50 ng) with or without S1P (1 μM), and network formation was assessed at 16 h by light microscopy (20× and 40×). (A) shows a representative image taken at 40× magnification, whereas (B) shows quantitation of the endothelial polygonal areas formed using 20× magnification. Bars, 50 μm (A). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to un-stimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (C) HPAECs processed as described in (B) were seeded on the top surface of Matrigel-coated polycarbonate membrane inserts of Boyden chamber. Cells were incubated with basal media along with indicated ligands (50 ng VEGF or 1 μM S1P) placed outside the chambers. After 16 h, ECs migrating to the bottom surface of the insert were fixed and nuclei were stained with DAPI, and their migration was determined using confocal microscope (20×). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to unstimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (D) Tumor growth in S1pr1 fl/fl versus EC-S1pr1 −/− mice following subcutaneous injection of 0.25 × 10 6 Lewis lung cell carcinoma (LLC) cells. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to S1pr1 fl/fl mice. Data expressed as mean ± SEM. (E) Tumor weight post day 21 of LLC cell injection in indicated strains of mice. Inset: a representative image of tumor from each strain. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 compared to S1pr1 fl/fl tumors. (F–H) S1pr1 fl/fl and EC-S1pr1 −/− tumor-associated ECs (TAECs) were isolated using FACS and cultured, and network formation and migration were assessed as described in (A)–(C). (F) shows the representative images of network formation; (G) shows the quantification of number of branch points formed; and (H) shows the quantification of the migrated cells per field. Bars, 50 μm. Image representative of multiple samples from different mice (F). Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs (G and H). All data represented were repeated multiple times and are expressed as mean ± SEM.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A and B) After 24 h post transfection of HPAECs with either scrambled siRNA (siScr) or S1PR1 siRNA (siS1PR1), these cells were transduced with control vector or hemagglutinin (HA)-tagged S1PR1 cDNA to restore S1PR1 expression. Cells were stimulated with VEGF (50 ng) with or without S1P (1 μM), and network formation was assessed at 16 h by light microscopy (20× and 40×). (A) shows a representative image taken at 40× magnification, whereas (B) shows quantitation of the endothelial polygonal areas formed using 20× magnification. Bars, 50 μm (A). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to un-stimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (C) HPAECs processed as described in (B) were seeded on the top surface of Matrigel-coated polycarbonate membrane inserts of Boyden chamber. Cells were incubated with basal media along with indicated ligands (50 ng VEGF or 1 μM S1P) placed outside the chambers. After 16 h, ECs migrating to the bottom surface of the insert were fixed and nuclei were stained with DAPI, and their migration was determined using confocal microscope (20×). One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared to unstimulated siScr, siS1PR1, and siS1PR1+ HA-S1PR1-cDNA-transfected HPAECs. ###p < 0.001 and ##p < 0.01 relative to siS1PR1-transfected HPAECs. (D) Tumor growth in S1pr1 fl/fl versus EC-S1pr1 −/− mice following subcutaneous injection of 0.25 × 10 6 Lewis lung cell carcinoma (LLC) cells. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to S1pr1 fl/fl mice. Data expressed as mean ± SEM. (E) Tumor weight post day 21 of LLC cell injection in indicated strains of mice. Inset: a representative image of tumor from each strain. n = 8 mice per group. Paired t test, two-tailed, ***p < 0.001 compared to S1pr1 fl/fl tumors. (F–H) S1pr1 fl/fl and EC-S1pr1 −/− tumor-associated ECs (TAECs) were isolated using FACS and cultured, and network formation and migration were assessed as described in (A)–(C). (F) shows the representative images of network formation; (G) shows the quantification of number of branch points formed; and (H) shows the quantification of the migrated cells per field. Bars, 50 μm. Image representative of multiple samples from different mice (F). Paired t test, two-tailed, ***p < 0.001 relative to S1PR1+ tumor ECs (G and H). All data represented were repeated multiple times and are expressed as mean ± SEM.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Transfection, Transduction, Plasmid Preparation, Expressing, Light Microscopy, Quantitation Assay, Two Tailed Test, Incubation, Staining, Migration, Microscopy, Injection, Isolation, Cell Culture

    (A) Schematic showing generation of mice for lineage tracing ECs using Floxed or S1PR1 fl / scl-creERT (5′ endothelial-cell-specific Scl-Cre ERT promotor) and Rosa26-tdTomato mice line. Tamoxifen injection induces Cre activity in the lox-P flanked locus, resulting in S1PR1 deletion in endothelial cells at stop sequence, turning these cells and their progeny to be labeled by tdTomato. (B and C) Indicated mice were injected with LLC cells as described in , and at 21 days, animals were injected with FITC-IB4 (intravenous [i.v.], 50 μg/100 μL) 4 h prior to sacrificing. Tumors were excised, stained with DAPI, and analyzed for tdTomato co-localization with IB4-positive vessels by using confocal microscope. (A) shows a representative image and (B) shows quantitation of vessel density. Bars, 100 μm. n = 5 mice per group. Paired t test, two-tailed, ***p < 0.001 relative to tdTomato-EC mice. (D and E) Indicated mice were treated either with VEGFR2 inhibitor (SU5416, 25 mg/kg) or S1PR1 inhibitor (R(W)146, 100 mg/kg) or a combination of both compounds. Mice received 8 doses of each compound, every 48 h beginning day 5 post LLC injection. Tumor volume was assessed using calipers every 48 h. n = 5 mice per group. One-way ANOVA, p < 0.05 (D). Post hoc Tukey’s test, ***p < 0.01 compared to R(W)146 compound, SU5416, and the combination of R(W) 146 and SU5416 injected group (D). One-way ANOVA, p > 0.05 (E). (F) TAECs seeded on poly-carbonate membrane were left untreated or treated either with SU5416 (100 μM) or R(W)146 (100 μM) or both for 2 h, and migration of tumor ECs was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to untreated S1PR1 + TAECs. #p < 0.05 compared to R(W)146-treated S1PR1 + TAECs. NS, not significantly different. All data expressed as mean ± SEM.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A) Schematic showing generation of mice for lineage tracing ECs using Floxed or S1PR1 fl / scl-creERT (5′ endothelial-cell-specific Scl-Cre ERT promotor) and Rosa26-tdTomato mice line. Tamoxifen injection induces Cre activity in the lox-P flanked locus, resulting in S1PR1 deletion in endothelial cells at stop sequence, turning these cells and their progeny to be labeled by tdTomato. (B and C) Indicated mice were injected with LLC cells as described in , and at 21 days, animals were injected with FITC-IB4 (intravenous [i.v.], 50 μg/100 μL) 4 h prior to sacrificing. Tumors were excised, stained with DAPI, and analyzed for tdTomato co-localization with IB4-positive vessels by using confocal microscope. (A) shows a representative image and (B) shows quantitation of vessel density. Bars, 100 μm. n = 5 mice per group. Paired t test, two-tailed, ***p < 0.001 relative to tdTomato-EC mice. (D and E) Indicated mice were treated either with VEGFR2 inhibitor (SU5416, 25 mg/kg) or S1PR1 inhibitor (R(W)146, 100 mg/kg) or a combination of both compounds. Mice received 8 doses of each compound, every 48 h beginning day 5 post LLC injection. Tumor volume was assessed using calipers every 48 h. n = 5 mice per group. One-way ANOVA, p < 0.05 (D). Post hoc Tukey’s test, ***p < 0.01 compared to R(W)146 compound, SU5416, and the combination of R(W) 146 and SU5416 injected group (D). One-way ANOVA, p > 0.05 (E). (F) TAECs seeded on poly-carbonate membrane were left untreated or treated either with SU5416 (100 μM) or R(W)146 (100 μM) or both for 2 h, and migration of tumor ECs was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 relative to untreated S1PR1 + TAECs. #p < 0.05 compared to R(W)146-treated S1PR1 + TAECs. NS, not significantly different. All data expressed as mean ± SEM.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Injection, Activity Assay, Sequencing, Labeling, Staining, Microscopy, Quantitation Assay, Two Tailed Test, Migration

    (A) Schematic showing generation of the S1PR1-GFP signaling mice . In the S1pr1 knock-in signaling mouse, S1PR1 is fused with two fusion proteins, namely, a tetracycline-regulated transactivator (tTA) and tobacco etch virus (TEV) protease along with β-arrestin. The S1pr1 knock-in signaling mouse is bred with H2B-GFP mouse to create the S1pr1-GFP signaling mouse where GFP expression reports for S1PR1 activity. (B and C) LLC cells were injected into H2B-GFP and S1PR1-GFP mice as described in . At day 16, tumors from these mice were harvested, sectioned, and stained with CD31 antibody to assess vessel density. (B) shows a representative image and (C) shows quantification of GFP+ vessel density based on CD31 staining. GFP signal serves as a proxy for S1P released by tumor cells and thereby the activation of S1PR1 in ECs. Note, a 5-fold magnified image of marked area in extreme right (B) shows S1PR1 activity in (GFP+) ECs (CD31+). Bar, 100 μm. Inset, indicates the area that was selected for magnification (5×) and presented at the right panel. n = 5 mice per group. Paired Student’s t test, ***p < 0.001 compared with H2B-GFP control. (D) qPCR of indicated genes in TAECs using specific primers. GAPDH expression was used as the internal control. One-way ANOVA for all genes. SPHK1, p < 0.05; SPHK2, p > 0.05; VEGF, p < 0.05; SPNS2, p < 0.05; GAPDH, p > 0.05. Paired Student’s t test, *p < 0.05 compared with LLCs. (E) LLCs were transfected with either scrambled siRNA (siScr) or Sphk1 (siSphk1) or Sphk2 (siSphk2) siRNA for 72 h. S1P concentrations were measured in indicated cells by using ELISA. One-way ANOVA, p < 0.01. Paired t test, two-tailed, ***p < 0.001 relative to siScr. **p < 0.01 relative to siScr. All data are representative of mean ± SEM from three individual experiments.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A) Schematic showing generation of the S1PR1-GFP signaling mice . In the S1pr1 knock-in signaling mouse, S1PR1 is fused with two fusion proteins, namely, a tetracycline-regulated transactivator (tTA) and tobacco etch virus (TEV) protease along with β-arrestin. The S1pr1 knock-in signaling mouse is bred with H2B-GFP mouse to create the S1pr1-GFP signaling mouse where GFP expression reports for S1PR1 activity. (B and C) LLC cells were injected into H2B-GFP and S1PR1-GFP mice as described in . At day 16, tumors from these mice were harvested, sectioned, and stained with CD31 antibody to assess vessel density. (B) shows a representative image and (C) shows quantification of GFP+ vessel density based on CD31 staining. GFP signal serves as a proxy for S1P released by tumor cells and thereby the activation of S1PR1 in ECs. Note, a 5-fold magnified image of marked area in extreme right (B) shows S1PR1 activity in (GFP+) ECs (CD31+). Bar, 100 μm. Inset, indicates the area that was selected for magnification (5×) and presented at the right panel. n = 5 mice per group. Paired Student’s t test, ***p < 0.001 compared with H2B-GFP control. (D) qPCR of indicated genes in TAECs using specific primers. GAPDH expression was used as the internal control. One-way ANOVA for all genes. SPHK1, p < 0.05; SPHK2, p > 0.05; VEGF, p < 0.05; SPNS2, p < 0.05; GAPDH, p > 0.05. Paired Student’s t test, *p < 0.05 compared with LLCs. (E) LLCs were transfected with either scrambled siRNA (siScr) or Sphk1 (siSphk1) or Sphk2 (siSphk2) siRNA for 72 h. S1P concentrations were measured in indicated cells by using ELISA. One-way ANOVA, p < 0.01. Paired t test, two-tailed, ***p < 0.001 relative to siScr. **p < 0.01 relative to siScr. All data are representative of mean ± SEM from three individual experiments.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Knock-In, Expressing, Activity Assay, Injection, Staining, Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    (A and B) FACS analysis of TAECs stained with anti-S1PR1, anti-VEGFR2, or control IgG antibodies and appropriate fluorescent-tagged secondary antibodies without permeabilization. Representative FACS profiles are presented in (A), and (B) shows quantitation of S1PR1- and VEGFR2-positive cells. Experiments were repeated three times. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (B). (C and D) Analysis of S1PR1 and VEGFR2 expression in S1pr1 +/+ and S1pr1 −/− TAECs by using confocal analysis. After fixing, TAECs were permeabilized and stained with indicated antibodies, followed by Alexa fluor-labeled secondary antibodies. Images were acquired using a confocal microscope. Bar, 10 μm. (C) shows images representative of experiments that were conducted multiple times, and in (D), the quantification of surface expression of VEGFR2 normalized to the expression of total VEGFR2 is represented by considering 20 cells. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (D). (E and F) HEK cells co-transfected with FLAG-VEGFR2 cDNA along with empty vector or HA-S1PR1 cDNA were biotinylated and then stimulated with 50 ng VEGF for indicated times. Total and cell surface expression of VEGFR2 and S1PR1 was determined using anti-VEGFR2 and anti-S1PR1 antibodies. A representative immunoblot is shown in (E), and (F) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, *p < 0.05 relative to cells transfected with VEGFR2 cDNA alone post without or 5 min VEGF stimulation and relative to cells transfected with VEGFR2 plus S1PR1 cDNA post 0–60 min of VEGF stimulation (E). UD, undetectable. Immunoblot is representative of three individual experiments. (G) At day 21, LLC tumor-bearing tdTomato-EC and tdTomato-EC-S1pr1 −/− mice were injected with FITC-IB4 (50 μg/100 μL, i.v). Tumors were harvested after 4 h, sectioned, and stained with anti-VEGFR2 antibody and DAPI. Tumor vessels were visualized using a confocal microscope. Right panel shows a 5-fold magnified area of marked tdTomato-positive vessel showing co-localizing of VEGFR2 with IB4. Bar, 50 μm. n = 5mice/group. All data expressed as mean ± SEM.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A and B) FACS analysis of TAECs stained with anti-S1PR1, anti-VEGFR2, or control IgG antibodies and appropriate fluorescent-tagged secondary antibodies without permeabilization. Representative FACS profiles are presented in (A), and (B) shows quantitation of S1PR1- and VEGFR2-positive cells. Experiments were repeated three times. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (B). (C and D) Analysis of S1PR1 and VEGFR2 expression in S1pr1 +/+ and S1pr1 −/− TAECs by using confocal analysis. After fixing, TAECs were permeabilized and stained with indicated antibodies, followed by Alexa fluor-labeled secondary antibodies. Images were acquired using a confocal microscope. Bar, 10 μm. (C) shows images representative of experiments that were conducted multiple times, and in (D), the quantification of surface expression of VEGFR2 normalized to the expression of total VEGFR2 is represented by considering 20 cells. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs (D). (E and F) HEK cells co-transfected with FLAG-VEGFR2 cDNA along with empty vector or HA-S1PR1 cDNA were biotinylated and then stimulated with 50 ng VEGF for indicated times. Total and cell surface expression of VEGFR2 and S1PR1 was determined using anti-VEGFR2 and anti-S1PR1 antibodies. A representative immunoblot is shown in (E), and (F) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, *p < 0.05 relative to cells transfected with VEGFR2 cDNA alone post without or 5 min VEGF stimulation and relative to cells transfected with VEGFR2 plus S1PR1 cDNA post 0–60 min of VEGF stimulation (E). UD, undetectable. Immunoblot is representative of three individual experiments. (G) At day 21, LLC tumor-bearing tdTomato-EC and tdTomato-EC-S1pr1 −/− mice were injected with FITC-IB4 (50 μg/100 μL, i.v). Tumors were harvested after 4 h, sectioned, and stained with anti-VEGFR2 antibody and DAPI. Tumor vessels were visualized using a confocal microscope. Right panel shows a 5-fold magnified area of marked tdTomato-positive vessel showing co-localizing of VEGFR2 with IB4. Bar, 50 μm. n = 5mice/group. All data expressed as mean ± SEM.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Staining, Quantitation Assay, Two Tailed Test, Expressing, Labeling, Microscopy, Transfection, Plasmid Preparation, Western Blot, Injection

    (A and B) Rac1 activity in control or S1PR1-depleted HPAECs following stimulation with 50 ng VEGF by using glutathione S-transferase (GST-tagged) PAK-PBD fusion protein and anti-Rac1 monoclonal antibody. Total cell lysates were immunoblotted with Rac1 or S1PR1 antibody. Representative immunoblot in (A), and (B) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 compared with siScr-transfected cells post 0, 10, and 30 min VEGF stimulation. *p < 0.05 compared with siScr post 0 or 10 min VEGF stimulation or siS1PR1-transfected cells post 0, 10, and 30 min VEGF stimulation. ###p < 0.001 and ##p < 0.01 relative to siScr-transfected cells post 0, 10, or 120 min VEGF stimulation. $$ p < 0.01 relative to siS1PR1-transfected cells post 60–120 min VEGF stimulation. (C and D) Rac1 activity in indicted ECs after treatment without or with VEGFR2 inhibitor SU5416 (100 μM) for 2 h. (C) shows the representative blot of active Rac1, and (D) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared with siScr 0 min VEGF stimulation of control treated group. ##p < 0.01 compared with siScr 60 min VEGF stimulation of control treated group. UD, undetectable. (E and F) Rac1 activity in indicated TAECs determined as described in (A). Total cell lysates were probed with S1PR1, VEGF2, and Rac1 antibodies. A representative blot is shown in (E), and (F) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs. (G) Migration of TAECs post treatment with indicated concentrations of NSC23766, a specific Rac1 inhibitor, determined as described in . One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with S1pr1 +/+ TAECs following treatment with 0, 0.1, or 1.0 μM Rac1 inhibitor; NS, compared with S1pr −/− TAECs following treatment with 0, 0.1, 1.0, or 10 μM Rac1 inhibitor. Data are representative of multiple experiments expressed as mean ± SEM. (H and I) HPAECs transfected with control (siScr) or Tiam1 siRNA for 48 h were stimulated with 50 ng VEGF, and Rac1 activity was determined as in (A). A representative blot is shown in (H), and (I) shows densitometry. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to unstimulated siScr; ##p < 0.01 relative to un-stimulated siScr transfected ECs; $$ p < 0.01 relative to siTiam1-transfected cells post 10 and 60 min VEGF stimulation. All immunoblots are representative of data from three individual experiments expressed as mean ± SEM.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A and B) Rac1 activity in control or S1PR1-depleted HPAECs following stimulation with 50 ng VEGF by using glutathione S-transferase (GST-tagged) PAK-PBD fusion protein and anti-Rac1 monoclonal antibody. Total cell lysates were immunoblotted with Rac1 or S1PR1 antibody. Representative immunoblot in (A), and (B) shows densitometry. One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 compared with siScr-transfected cells post 0, 10, and 30 min VEGF stimulation. *p < 0.05 compared with siScr post 0 or 10 min VEGF stimulation or siS1PR1-transfected cells post 0, 10, and 30 min VEGF stimulation. ###p < 0.001 and ##p < 0.01 relative to siScr-transfected cells post 0, 10, or 120 min VEGF stimulation. $$ p < 0.01 relative to siS1PR1-transfected cells post 60–120 min VEGF stimulation. (C and D) Rac1 activity in indicted ECs after treatment without or with VEGFR2 inhibitor SU5416 (100 μM) for 2 h. (C) shows the representative blot of active Rac1, and (D) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 and **p < 0.01 compared with siScr 0 min VEGF stimulation of control treated group. ##p < 0.01 compared with siScr 60 min VEGF stimulation of control treated group. UD, undetectable. (E and F) Rac1 activity in indicated TAECs determined as described in (A). Total cell lysates were probed with S1PR1, VEGF2, and Rac1 antibodies. A representative blot is shown in (E), and (F) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + TAECs. (G) Migration of TAECs post treatment with indicated concentrations of NSC23766, a specific Rac1 inhibitor, determined as described in . One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with S1pr1 +/+ TAECs following treatment with 0, 0.1, or 1.0 μM Rac1 inhibitor; NS, compared with S1pr −/− TAECs following treatment with 0, 0.1, 1.0, or 10 μM Rac1 inhibitor. Data are representative of multiple experiments expressed as mean ± SEM. (H and I) HPAECs transfected with control (siScr) or Tiam1 siRNA for 48 h were stimulated with 50 ng VEGF, and Rac1 activity was determined as in (A). A representative blot is shown in (H), and (I) shows densitometry. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to unstimulated siScr; ##p < 0.01 relative to un-stimulated siScr transfected ECs; $$ p < 0.01 relative to siTiam1-transfected cells post 10 and 60 min VEGF stimulation. All immunoblots are representative of data from three individual experiments expressed as mean ± SEM.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Activity Assay, Western Blot, Two Tailed Test, Transfection, Migration

    (A and B) VEGFR2 phosphorylation at Y951 or Y1175 in S1pr1 +/+ or S1pr1 −/− TAECs determined using site-specific phosphoVEGFR2 antibodies and total VEGFR2 and S1PR1 antibodies. A representative immunoblot is shown in (A), and (B) shows densitometry. Paired t test, two-tailed, ***p < 0.001 and *p < 0.05 relative to S1pr1 +/+ TAECs. (C–E) Phosphorylation of VEGFR2 in control or S1PR1-depleted HPAECs determined as described in (A) and (B). (C) shows the representative immunoblot of VEGFR2 phosphorylation at Y1175 and Y951 and total VEGFR2, and (D) and (E) show the indicated densitometries. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0 min VEGF stimulation (D). Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0, 10, 20, 60, and 120 min VEGF stimulation (E). (F and G) Y951-VEGFR2 phosphorylation in control or S1PR1-depleted HPAECs after 2 h treatment without or with SU5416. (F) shows the representative immunoblot, and (G) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to siScr control 0 min VEGF stimulated. ##p < 0.01 compared to control siS1PR1 0 and 60 min VEGF stimulation. (H and I) S1pr1 +/+ or S1pr1 −/− TAECs were treated with control anti-mouse IgG or anti-mouse-VEGF-A antibody (0.5 mg/ml) for 2h, after which VEGFR2 phosphorylation at Y951 or Y1175 and total VEGFR2 expression were determined. (H) shows a representative immunoblot, and (I) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, *** and ###p < 0.001 relative to S1pr1 +/+ TAECs following VEGF-A antibody treatment. (J) HPAECs transfected with vector, VEGFR2 cDNA, or phosphor-defective VEGFR2 cDNA (Y951F-VEGFR2 mutant) for 24 h were seeded on the polycarbonate membrane, and EC migration was determined as in . One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 relative to un-stimulated vector or VEGFR2 or Y951F-VEGFR2 cDNA transfected cells. ###p < 0.001 relative to stimulated Y951F-VEGFR2-mutant-transfected cells, **p < 0.01 relative to unstimulated VEGFR2 or Y951-VEGFR2 transfected cells. Data representative of mean ± SEM from three independent experiments. (K–N) Phosphorylation of VEGFR2 at Y1175/Y951 and Rac1 activity in HPAECs transfected with indicated mutants. (K) shows the representative immunoblotting images, and (L)–(N) show the indicated densitometries. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2-transfected cells (L). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 transfected cells. ##p < 0.01 compared to 0, 10, and 60 min VEGF-stimulated Y951F VEGFR2-transfected cells (M). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 or Y951F VEGFR2 transfected cells (N). All immunoblots represent data from three individual experiments expressed as mean ± SEM.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A and B) VEGFR2 phosphorylation at Y951 or Y1175 in S1pr1 +/+ or S1pr1 −/− TAECs determined using site-specific phosphoVEGFR2 antibodies and total VEGFR2 and S1PR1 antibodies. A representative immunoblot is shown in (A), and (B) shows densitometry. Paired t test, two-tailed, ***p < 0.001 and *p < 0.05 relative to S1pr1 +/+ TAECs. (C–E) Phosphorylation of VEGFR2 in control or S1PR1-depleted HPAECs determined as described in (A) and (B). (C) shows the representative immunoblot of VEGFR2 phosphorylation at Y1175 and Y951 and total VEGFR2, and (D) and (E) show the indicated densitometries. One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0 min VEGF stimulation (D). Paired t test, two-tailed, **p < 0.01 compared with siScr 0 min VEGF stimulation. ##p < 0.01 compared with siS1PR1 0, 10, 20, 60, and 120 min VEGF stimulation (E). (F and G) Y951-VEGFR2 phosphorylation in control or S1PR1-depleted HPAECs after 2 h treatment without or with SU5416. (F) shows the representative immunoblot, and (G) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to siScr control 0 min VEGF stimulated. ##p < 0.01 compared to control siS1PR1 0 and 60 min VEGF stimulation. (H and I) S1pr1 +/+ or S1pr1 −/− TAECs were treated with control anti-mouse IgG or anti-mouse-VEGF-A antibody (0.5 mg/ml) for 2h, after which VEGFR2 phosphorylation at Y951 or Y1175 and total VEGFR2 expression were determined. (H) shows a representative immunoblot, and (I) shows densitometry. One-way ANOVA, p < 0.001. Paired t test, two-tailed, *** and ###p < 0.001 relative to S1pr1 +/+ TAECs following VEGF-A antibody treatment. (J) HPAECs transfected with vector, VEGFR2 cDNA, or phosphor-defective VEGFR2 cDNA (Y951F-VEGFR2 mutant) for 24 h were seeded on the polycarbonate membrane, and EC migration was determined as in . One-way ANOVA, p < 0.001. Paired t test, two-tailed, ***p < 0.001 relative to un-stimulated vector or VEGFR2 or Y951F-VEGFR2 cDNA transfected cells. ###p < 0.001 relative to stimulated Y951F-VEGFR2-mutant-transfected cells, **p < 0.01 relative to unstimulated VEGFR2 or Y951-VEGFR2 transfected cells. Data representative of mean ± SEM from three independent experiments. (K–N) Phosphorylation of VEGFR2 at Y1175/Y951 and Rac1 activity in HPAECs transfected with indicated mutants. (K) shows the representative immunoblotting images, and (L)–(N) show the indicated densitometries. One-way ANOVA, p < 0.001. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2-transfected cells (L). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 transfected cells. ##p < 0.01 compared to 0, 10, and 60 min VEGF-stimulated Y951F VEGFR2-transfected cells (M). One-way ANOVA, p < 0.01. Paired t test, two-tailed, **p < 0.01 compared to un-stimulated vector or VEGFR2 or Y951F VEGFR2 transfected cells (N). All immunoblots represent data from three individual experiments expressed as mean ± SEM.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Western Blot, Two Tailed Test, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Migration, Activity Assay

    (A–D) HPAECs treated without or with pertussis toxin (PTX; 50 μM) were stimulated with VEGF for indicated time points. Phosphorylation of VEGFR2 and c-Abl1 was determined using phosphospecific antibodies. Immunoblot with c-Abl1 and VEGFR2 expression was used as loading control. (A) shows a representative immunoblot, and (B)–(D) show densitometry. One-way ANOVA, p < 0.05 (B). One-way ANOVA, p < 0.001 (C and D). Paired t test, two-tailed, ***p < 0.001 relative to unstimulated HPAECs (B–D). (E and F) Lysate from S1pr1 +/+ or S1pr1 −/− TAECs were immunoprecipitated with anti-VEGFR2 antibody, and immunocomplexes were probed with c-Abl1 or VEGFR2 antibodies. A representative immunoblot is shown in (E), and (F) shows fold change in VEGFR2-c-Abl interaction normalized against total VEGFR2. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (G and H) Lysates from indicated TAECs were immunoblotted with either phospho-Src, phospho-c-Abl1, c-Src, or c-Abl1 antibodies. A representative immunoblot is shown in (G), and (H) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (I) Indicated TAECs seeded on polycarbonate membrane were treated with or without imatinib (40 μM for 1 h), and EC migration was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 relative to untreated S1PR1 + tumor ECs; NS relative to imatinib-treated S1PR1 + or S1PR1 − TAECs. Data representative of mean ± SEM from multiple experiments. (J–M) HPAECs transfected with c-Abl1 siRNA for 72 h were stimulated with VEGF for indicated time points. VEGFR2 phosphorylation and Rac1 activity were determined as described in . Immunoblotting with c-Abl antibody was used to determine c-Abl1 depletion. Total Rac1, total VEGFR2, and actin were used as loading control. A representative immunoblot is shown in (J), and (K)–(M) show densitometries. One-way ANOVA, p < 0.01 (K–M). Paired t test, two-tailed, **p < 0.001 relative to un-stimulated siScr or c-Abl-siRNA-transfected cells; ##p < 0.01 relative to siScr HPAECs following 10 and 120 min VEGF stimulation (K–M). All western blots show data from multiple experiments expressed as mean ± SEM. (N) Model showing influence of S1P-S1PR1 signaling on VEGF-VEGFR2-mediated angiogenesis that augments EC migration and tumor vascularization and growth. Cancer cells generate S1P and VEGF. S1P ligates S1PR1 in S1PR1+ ECs. Additionally, activation of SPHK induce S1PR1 activity in a paracrine manner. S1PR1, in turn, stimulates Gi, which promotes c-Abl1 activity by VEGF. Activated c-Abl1 phosphorylates VEGFR2 on Y951, reducing VEGFR2 binding with unknown endocytic protein, causing VEGFR2 to remain on the EC surface and prolonging Rac1 activity in a Tiam1-dependent manner that increases EC migration and efficient tumor vascularization. In ECs lacking S1PR1, VEGF leads to VEGFR2 phosphorylation at Y1175, followed by receptor internalization. Internalized receptor induces ERK activity but transiently activates Rac1, leading to reduced EC migration and impairing tumor vascularization and growth.

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: (A–D) HPAECs treated without or with pertussis toxin (PTX; 50 μM) were stimulated with VEGF for indicated time points. Phosphorylation of VEGFR2 and c-Abl1 was determined using phosphospecific antibodies. Immunoblot with c-Abl1 and VEGFR2 expression was used as loading control. (A) shows a representative immunoblot, and (B)–(D) show densitometry. One-way ANOVA, p < 0.05 (B). One-way ANOVA, p < 0.001 (C and D). Paired t test, two-tailed, ***p < 0.001 relative to unstimulated HPAECs (B–D). (E and F) Lysate from S1pr1 +/+ or S1pr1 −/− TAECs were immunoprecipitated with anti-VEGFR2 antibody, and immunocomplexes were probed with c-Abl1 or VEGFR2 antibodies. A representative immunoblot is shown in (E), and (F) shows fold change in VEGFR2-c-Abl interaction normalized against total VEGFR2. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (G and H) Lysates from indicated TAECs were immunoblotted with either phospho-Src, phospho-c-Abl1, c-Src, or c-Abl1 antibodies. A representative immunoblot is shown in (G), and (H) shows densitometry. Paired t test, two-tailed, ***p < 0.001 relative to S1PR1 + tumor ECs. (I) Indicated TAECs seeded on polycarbonate membrane were treated with or without imatinib (40 μM for 1 h), and EC migration was determined as described in . One-way ANOVA, p < 0.05. Paired t test, two-tailed, **p < 0.01 relative to untreated S1PR1 + tumor ECs; NS relative to imatinib-treated S1PR1 + or S1PR1 − TAECs. Data representative of mean ± SEM from multiple experiments. (J–M) HPAECs transfected with c-Abl1 siRNA for 72 h were stimulated with VEGF for indicated time points. VEGFR2 phosphorylation and Rac1 activity were determined as described in . Immunoblotting with c-Abl antibody was used to determine c-Abl1 depletion. Total Rac1, total VEGFR2, and actin were used as loading control. A representative immunoblot is shown in (J), and (K)–(M) show densitometries. One-way ANOVA, p < 0.01 (K–M). Paired t test, two-tailed, **p < 0.001 relative to un-stimulated siScr or c-Abl-siRNA-transfected cells; ##p < 0.01 relative to siScr HPAECs following 10 and 120 min VEGF stimulation (K–M). All western blots show data from multiple experiments expressed as mean ± SEM. (N) Model showing influence of S1P-S1PR1 signaling on VEGF-VEGFR2-mediated angiogenesis that augments EC migration and tumor vascularization and growth. Cancer cells generate S1P and VEGF. S1P ligates S1PR1 in S1PR1+ ECs. Additionally, activation of SPHK induce S1PR1 activity in a paracrine manner. S1PR1, in turn, stimulates Gi, which promotes c-Abl1 activity by VEGF. Activated c-Abl1 phosphorylates VEGFR2 on Y951, reducing VEGFR2 binding with unknown endocytic protein, causing VEGFR2 to remain on the EC surface and prolonging Rac1 activity in a Tiam1-dependent manner that increases EC migration and efficient tumor vascularization. In ECs lacking S1PR1, VEGF leads to VEGFR2 phosphorylation at Y1175, followed by receptor internalization. Internalized receptor induces ERK activity but transiently activates Rac1, leading to reduced EC migration and impairing tumor vascularization and growth.

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Western Blot, Expressing, Two Tailed Test, Immunoprecipitation, Migration, Transfection, Activity Assay, Activation Assay, Binding Assay

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling

    doi: 10.1016/j.celrep.2019.11.036

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Anti-Sphingosine 1-Phosphate Receptor 1 (extracellular) , Alamone labs , #ASR-011; RRID: AB_2039836.

    Techniques: Labeling, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Software

    Representative blots of S1PR1 levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG35–55–treated animals that received vehicle.

    Journal: Pain

    Article Title: Fingolimod Reduces Neuropathic Pain Behaviors in a Mouse Model of Multiple Sclerosis by a Sphingosine-1 Phosphate Receptor 1-Dependent Inhibition of Central Sensitization in the Dorsal Horn.

    doi: 10.1097/j.pain.0000000000001106

    Figure Lengend Snippet: Representative blots of S1PR1 levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG35–55–treated animals that received vehicle.

    Article Snippet: Lanes from each image were loaded with 20 mg protein. (A) Alomone #ASR-011 anti-Sphingosine 1-Phosphate Receptor 1 antibody (1:200).

    Techniques: Expressing

    AD2900 shows antagonistic activities against  S1P1,  2, 3, 4, and 5

    Journal: Oncotarget

    Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

    doi: 10.18632/oncotarget.18626

    Figure Lengend Snippet: AD2900 shows antagonistic activities against S1P1, 2, 3, 4, and 5

    Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

    Techniques:

    (A, B) The percentages of S1P1-positive PBMCs after the treatment with different concentrations of AD2900, FTY720, or SEW2871 and at different time points were examined by FACS analysis. S1P1 expression was tested in PBMCs after a 30-min treatment with AD2900 at different concentrations (A) or after a 30-min or 60-min treatment with 100 nM AD2900, FTY720, or SEW2871 (B) . (C) The percentage of CCR7-positive PBMCs was tested by FACS analysis after a 30-min treatment with 100 nM AD2900, FTY720, or SEW2871. All the significances are compared to untreated PBMCs. Results summarize the results of at least four independent experiments. Results of Student’s t -test: *(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

    Journal: Oncotarget

    Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

    doi: 10.18632/oncotarget.18626

    Figure Lengend Snippet: (A, B) The percentages of S1P1-positive PBMCs after the treatment with different concentrations of AD2900, FTY720, or SEW2871 and at different time points were examined by FACS analysis. S1P1 expression was tested in PBMCs after a 30-min treatment with AD2900 at different concentrations (A) or after a 30-min or 60-min treatment with 100 nM AD2900, FTY720, or SEW2871 (B) . (C) The percentage of CCR7-positive PBMCs was tested by FACS analysis after a 30-min treatment with 100 nM AD2900, FTY720, or SEW2871. All the significances are compared to untreated PBMCs. Results summarize the results of at least four independent experiments. Results of Student’s t -test: *(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

    Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

    Techniques: Expressing, Two Tailed Test

    C57BL/6 mice were orally administered with 1.8, 2.7, and 3.6 mg/l AD2900 or 1.8 mg/l FTY720 for 2 days, as shown in Figure . Leukocytes from blood, spleen, and pLNs were collected and stained with CD3e and S1P1 or CCR7 fluorescent antibodies and then analyzed by FACS analysis. The percentages of S1P1+ CD3e+ T cells from blood (A) , spleen (B) , and pLNs (C) are shown. The percentages of CCR7+ CD3e+ T cells from blood (D) , spleen (E) , and pLNs (F) are shown. All the significances are compared to untreated healthy mice. Results summarize at least three independent experiments. Results of Student’s t -test:*(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

    Journal: Oncotarget

    Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

    doi: 10.18632/oncotarget.18626

    Figure Lengend Snippet: C57BL/6 mice were orally administered with 1.8, 2.7, and 3.6 mg/l AD2900 or 1.8 mg/l FTY720 for 2 days, as shown in Figure . Leukocytes from blood, spleen, and pLNs were collected and stained with CD3e and S1P1 or CCR7 fluorescent antibodies and then analyzed by FACS analysis. The percentages of S1P1+ CD3e+ T cells from blood (A) , spleen (B) , and pLNs (C) are shown. The percentages of CCR7+ CD3e+ T cells from blood (D) , spleen (E) , and pLNs (F) are shown. All the significances are compared to untreated healthy mice. Results summarize at least three independent experiments. Results of Student’s t -test:*(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

    Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

    Techniques: Staining, Two Tailed Test

    As an antagonist to S1P receptors 1–5, AD2900 can compete with S1P to bind S1P receptors leading to reduced S1P signaling and enhanced expression of S1P1 on T cells in S1P-rich environments such as the blood and the spleen. This altered expression, together with decreased CCR7 expression, inhibits T-cell entry into the lymph nodes (LNs) from the blood, causing accumulation of T cells in the blood. However, the entry of T cells to the spleen is not affected because it is not S1P dependent. Since Tcm-like cells express CCR7, these cells are attracted to the spleen and accumulate in it; yet, S1P1 elevated expression may have an effect on the S1P-dependent ingression of these cells from the MZ to the white pulp. Tef/em-like cells, which are CCR7 negative, are the primary T-cell subpopulation in the blood after AD2900 treatment. The significant decrease in naive T-cell counts in the circulation and peripheral lymphoid tissues tested may be explained by the inhibition of S1P signaling in the thymus leading to attenuated T-cell egression from the thymus to the circulation. Arrow key: thick = response; dashed = inhibition.

    Journal: Oncotarget

    Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

    doi: 10.18632/oncotarget.18626

    Figure Lengend Snippet: As an antagonist to S1P receptors 1–5, AD2900 can compete with S1P to bind S1P receptors leading to reduced S1P signaling and enhanced expression of S1P1 on T cells in S1P-rich environments such as the blood and the spleen. This altered expression, together with decreased CCR7 expression, inhibits T-cell entry into the lymph nodes (LNs) from the blood, causing accumulation of T cells in the blood. However, the entry of T cells to the spleen is not affected because it is not S1P dependent. Since Tcm-like cells express CCR7, these cells are attracted to the spleen and accumulate in it; yet, S1P1 elevated expression may have an effect on the S1P-dependent ingression of these cells from the MZ to the white pulp. Tef/em-like cells, which are CCR7 negative, are the primary T-cell subpopulation in the blood after AD2900 treatment. The significant decrease in naive T-cell counts in the circulation and peripheral lymphoid tissues tested may be explained by the inhibition of S1P signaling in the thymus leading to attenuated T-cell egression from the thymus to the circulation. Arrow key: thick = response; dashed = inhibition.

    Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

    Techniques: Expressing, Inhibition