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s1p  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences s1p
    Selective inhibition of SphK2 with a pharmacological inhibitor inhibits tumor tissue nuclear bulk histone acetylation, HIFα, HIFα-target genes, and in vivo TNBC tumor growth. A) MCF-7 cells grown in low-attachment tissue culture plate for 7 days to form spheroids were treated with1 µM K-145 or vehicle for 2 days, as indicated. Light phase-contrast microscopy images were taken with 10X magnification; representative images are shown. B) Duplicate cultures were stained with LIVE/DEAD staining. For some experiments, nuclear proteins from MCF-7 spheroids treated with or without K-145 or naïve MCF-7 cells grown in tissue culture plates were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. D-G) Duplicate cultures of MCF-7 spheroids and cells were used for quantitative real-time PCR for the HIF-2α, VEGFA, and SphK2 gene levels normalized to GAPDH. Data are means ± SD, P<0.05 vs. control, by Student’s t-test. K-145 inhibited growth of MDA-MB-231 xenografts. H) MDA-MB-231 cells were surgically implanted under the fat pads of NSG mice. Mice bearing MDA-MB-231 tumors (~50–100 mm3) were separated into 2 groups randomly (N=5/group). Vehicle or K-145 (30 mg/Kg) was administered i.p for 35 days. Tumor volumes were recorded and calculated every 2–4 days until sacrifice as mm3 ± SD. *, P< 0.05 vs. vehicle, by Student’s t-test. Of note, visible breast cancer lung metastasis was not observed in our experimental setting with this xenograft model. I) Primary tumor weight was measured, data are means ± SD, P<0.05 vs. control. J) Tumor tissue nuclei were used for <t>S1P</t> and DH-S1P measurements using LC-MS/MS methods. K) Equal amounts of tumor tissue nuclear proteins were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. Histone H3 is used as a loading control for nuclear proteins. L-N) Tumor tissues treated with vehicle or K-145 were subjected to quantitative real-time PCR for the HIF-2α, VEGFA, and SERPINE1 gene levels normalized to GAPDH. Data are means ± SD. P<0.05 vs. vehicle.
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    Images

    1) Product Images from "Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate"

    Article Title: Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    doi: 10.1096/fj.201901734RR

    Selective inhibition of SphK2 with a pharmacological inhibitor inhibits tumor tissue nuclear bulk histone acetylation, HIFα, HIFα-target genes, and in vivo TNBC tumor growth. A) MCF-7 cells grown in low-attachment tissue culture plate for 7 days to form spheroids were treated with1 µM K-145 or vehicle for 2 days, as indicated. Light phase-contrast microscopy images were taken with 10X magnification; representative images are shown. B) Duplicate cultures were stained with LIVE/DEAD staining. For some experiments, nuclear proteins from MCF-7 spheroids treated with or without K-145 or naïve MCF-7 cells grown in tissue culture plates were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. D-G) Duplicate cultures of MCF-7 spheroids and cells were used for quantitative real-time PCR for the HIF-2α, VEGFA, and SphK2 gene levels normalized to GAPDH. Data are means ± SD, P<0.05 vs. control, by Student’s t-test. K-145 inhibited growth of MDA-MB-231 xenografts. H) MDA-MB-231 cells were surgically implanted under the fat pads of NSG mice. Mice bearing MDA-MB-231 tumors (~50–100 mm3) were separated into 2 groups randomly (N=5/group). Vehicle or K-145 (30 mg/Kg) was administered i.p for 35 days. Tumor volumes were recorded and calculated every 2–4 days until sacrifice as mm3 ± SD. *, P< 0.05 vs. vehicle, by Student’s t-test. Of note, visible breast cancer lung metastasis was not observed in our experimental setting with this xenograft model. I) Primary tumor weight was measured, data are means ± SD, P<0.05 vs. control. J) Tumor tissue nuclei were used for S1P and DH-S1P measurements using LC-MS/MS methods. K) Equal amounts of tumor tissue nuclear proteins were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. Histone H3 is used as a loading control for nuclear proteins. L-N) Tumor tissues treated with vehicle or K-145 were subjected to quantitative real-time PCR for the HIF-2α, VEGFA, and SERPINE1 gene levels normalized to GAPDH. Data are means ± SD. P<0.05 vs. vehicle.
    Figure Legend Snippet: Selective inhibition of SphK2 with a pharmacological inhibitor inhibits tumor tissue nuclear bulk histone acetylation, HIFα, HIFα-target genes, and in vivo TNBC tumor growth. A) MCF-7 cells grown in low-attachment tissue culture plate for 7 days to form spheroids were treated with1 µM K-145 or vehicle for 2 days, as indicated. Light phase-contrast microscopy images were taken with 10X magnification; representative images are shown. B) Duplicate cultures were stained with LIVE/DEAD staining. For some experiments, nuclear proteins from MCF-7 spheroids treated with or without K-145 or naïve MCF-7 cells grown in tissue culture plates were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. D-G) Duplicate cultures of MCF-7 spheroids and cells were used for quantitative real-time PCR for the HIF-2α, VEGFA, and SphK2 gene levels normalized to GAPDH. Data are means ± SD, P<0.05 vs. control, by Student’s t-test. K-145 inhibited growth of MDA-MB-231 xenografts. H) MDA-MB-231 cells were surgically implanted under the fat pads of NSG mice. Mice bearing MDA-MB-231 tumors (~50–100 mm3) were separated into 2 groups randomly (N=5/group). Vehicle or K-145 (30 mg/Kg) was administered i.p for 35 days. Tumor volumes were recorded and calculated every 2–4 days until sacrifice as mm3 ± SD. *, P< 0.05 vs. vehicle, by Student’s t-test. Of note, visible breast cancer lung metastasis was not observed in our experimental setting with this xenograft model. I) Primary tumor weight was measured, data are means ± SD, P<0.05 vs. control. J) Tumor tissue nuclei were used for S1P and DH-S1P measurements using LC-MS/MS methods. K) Equal amounts of tumor tissue nuclear proteins were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. Histone H3 is used as a loading control for nuclear proteins. L-N) Tumor tissues treated with vehicle or K-145 were subjected to quantitative real-time PCR for the HIF-2α, VEGFA, and SERPINE1 gene levels normalized to GAPDH. Data are means ± SD. P<0.05 vs. vehicle.

    Techniques Used: Inhibition, In Vivo, Microscopy, Staining, Western Blot, Real-time Polymerase Chain Reaction, Control, Liquid Chromatography with Mass Spectroscopy

    SphK2 enhances HIF-1α binding to the VEGF promoter. MCF-7 cells (A, B) or MDA-MB-231 cells (C) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or normoxia (white bar) for 40 min. For some experiments, MCF-7 cells transfected with vector (light gray bar) or V5-SphK2 (dark gray bar) were incubated at normoxia (C). Cells were subjected to ChIP analyses with antibodies to HIF-1α or normal IgG. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the VEGFA gene (A, C). For some experiments, ChIP-qPCR products were analyzed on a 2% Agarose gel (B). Relative binding to the VEGFA promoter is expressed as the fold enriched of input. Data are means ± SD. P < 0.05, relative to siControl (A) or vector transfectants (C), by Student’s t-test. D-H) MDA-MB-231 cells transfected with siControl or siSphK2 were treated with hypoxia (black bar) or normoxia (white bar) for 40 min and subjected to qPCR analyses with the specific primers to human SERPINE (D), OCT4 (E), ALKBH5 (F), CD44 (G), and VEGFA (H). mRNA levels of the genes, as indicated, were determined by quantitative real-time PCR and normalized to GAPDH. Data are means ± SD, P<0.05 vs. siControl, by Student’s t-test. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα-direct target gene expression by hypoxia.
    Figure Legend Snippet: SphK2 enhances HIF-1α binding to the VEGF promoter. MCF-7 cells (A, B) or MDA-MB-231 cells (C) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or normoxia (white bar) for 40 min. For some experiments, MCF-7 cells transfected with vector (light gray bar) or V5-SphK2 (dark gray bar) were incubated at normoxia (C). Cells were subjected to ChIP analyses with antibodies to HIF-1α or normal IgG. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the VEGFA gene (A, C). For some experiments, ChIP-qPCR products were analyzed on a 2% Agarose gel (B). Relative binding to the VEGFA promoter is expressed as the fold enriched of input. Data are means ± SD. P < 0.05, relative to siControl (A) or vector transfectants (C), by Student’s t-test. D-H) MDA-MB-231 cells transfected with siControl or siSphK2 were treated with hypoxia (black bar) or normoxia (white bar) for 40 min and subjected to qPCR analyses with the specific primers to human SERPINE (D), OCT4 (E), ALKBH5 (F), CD44 (G), and VEGFA (H). mRNA levels of the genes, as indicated, were determined by quantitative real-time PCR and normalized to GAPDH. Data are means ± SD, P<0.05 vs. siControl, by Student’s t-test. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα-direct target gene expression by hypoxia.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Targeted Gene Expression

    SphK2 enhances HIF-1α protein binding to the HIF-2α promoter. MDA-MB-231 cells (A, B) or MCF7 cells (E, F) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or left at normoxia (white bar) for 40 min and subjected to ChIP analyses with antibodies to HIF-1α or normal rabbit IgG, as indicated. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the HIF-2α gene. ChIP-qPCR products for HIF-2α were analyzed on a 2% Agarose gel (B, F). MDA-MB-231 cells (C, D) or MCF-7 cells (G, H) were transfected with vector (light gray) or V5-SphK2 (dark gray) and processed for ChIP analyses with antibodies to HIF-1α or normal control IgG. ChIP-PCR products were analyzed on a 2% Agarose gel, and representative images are shown (D, H). Relative binding to the promoter using ChIP-qPCR data is expressed as the fold enriched of input. Data are means ± SD, P<0.05, relative to siControl (A, E), or vector transfectants (C, G), respectively. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα positive feedback regulation by hypoxia.
    Figure Legend Snippet: SphK2 enhances HIF-1α protein binding to the HIF-2α promoter. MDA-MB-231 cells (A, B) or MCF7 cells (E, F) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or left at normoxia (white bar) for 40 min and subjected to ChIP analyses with antibodies to HIF-1α or normal rabbit IgG, as indicated. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the HIF-2α gene. ChIP-qPCR products for HIF-2α were analyzed on a 2% Agarose gel (B, F). MDA-MB-231 cells (C, D) or MCF-7 cells (G, H) were transfected with vector (light gray) or V5-SphK2 (dark gray) and processed for ChIP analyses with antibodies to HIF-1α or normal control IgG. ChIP-PCR products were analyzed on a 2% Agarose gel, and representative images are shown (D, H). Relative binding to the promoter using ChIP-qPCR data is expressed as the fold enriched of input. Data are means ± SD, P<0.05, relative to siControl (A, E), or vector transfectants (C, G), respectively. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα positive feedback regulation by hypoxia.

    Techniques Used: Protein Binding, Transfection, Real-time Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation, Control, Binding Assay

    Nuclear S1P directly binds to HIFα and promotes activation in response to hypoxia.
    Figure Legend Snippet: Nuclear S1P directly binds to HIFα and promotes activation in response to hypoxia.

    Techniques Used: Activation Assay



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    Selective inhibition of SphK2 with a pharmacological inhibitor inhibits tumor tissue nuclear bulk histone acetylation, HIFα, HIFα-target genes, and in vivo TNBC tumor growth. A) MCF-7 cells grown in low-attachment tissue culture plate for 7 days to form spheroids were treated with1 µM K-145 or vehicle for 2 days, as indicated. Light phase-contrast microscopy images were taken with 10X magnification; representative images are shown. B) Duplicate cultures were stained with LIVE/DEAD staining. For some experiments, nuclear proteins from MCF-7 spheroids treated with or without K-145 or naïve MCF-7 cells grown in tissue culture plates were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. D-G) Duplicate cultures of MCF-7 spheroids and cells were used for quantitative real-time PCR for the HIF-2α, VEGFA, and SphK2 gene levels normalized to GAPDH. Data are means ± SD, P<0.05 vs. control, by Student’s t-test. K-145 inhibited growth of MDA-MB-231 xenografts. H) MDA-MB-231 cells were surgically implanted under the fat pads of NSG mice. Mice bearing MDA-MB-231 tumors (~50–100 mm3) were separated into 2 groups randomly (N=5/group). Vehicle or K-145 (30 mg/Kg) was administered i.p for 35 days. Tumor volumes were recorded and calculated every 2–4 days until sacrifice as mm3 ± SD. *, P< 0.05 vs. vehicle, by Student’s t-test. Of note, visible breast cancer lung metastasis was not observed in our experimental setting with this xenograft model. I) Primary tumor weight was measured, data are means ± SD, P<0.05 vs. control. J) Tumor tissue nuclei were used for S1P and DH-S1P measurements using LC-MS/MS methods. K) Equal amounts of tumor tissue nuclear proteins were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. Histone H3 is used as a loading control for nuclear proteins. L-N) Tumor tissues treated with vehicle or K-145 were subjected to quantitative real-time PCR for the HIF-2α, VEGFA, and SERPINE1 gene levels normalized to GAPDH. Data are means ± SD. P<0.05 vs. vehicle.

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    Article Title: Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate

    doi: 10.1096/fj.201901734RR

    Figure Lengend Snippet: Selective inhibition of SphK2 with a pharmacological inhibitor inhibits tumor tissue nuclear bulk histone acetylation, HIFα, HIFα-target genes, and in vivo TNBC tumor growth. A) MCF-7 cells grown in low-attachment tissue culture plate for 7 days to form spheroids were treated with1 µM K-145 or vehicle for 2 days, as indicated. Light phase-contrast microscopy images were taken with 10X magnification; representative images are shown. B) Duplicate cultures were stained with LIVE/DEAD staining. For some experiments, nuclear proteins from MCF-7 spheroids treated with or without K-145 or naïve MCF-7 cells grown in tissue culture plates were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. D-G) Duplicate cultures of MCF-7 spheroids and cells were used for quantitative real-time PCR for the HIF-2α, VEGFA, and SphK2 gene levels normalized to GAPDH. Data are means ± SD, P<0.05 vs. control, by Student’s t-test. K-145 inhibited growth of MDA-MB-231 xenografts. H) MDA-MB-231 cells were surgically implanted under the fat pads of NSG mice. Mice bearing MDA-MB-231 tumors (~50–100 mm3) were separated into 2 groups randomly (N=5/group). Vehicle or K-145 (30 mg/Kg) was administered i.p for 35 days. Tumor volumes were recorded and calculated every 2–4 days until sacrifice as mm3 ± SD. *, P< 0.05 vs. vehicle, by Student’s t-test. Of note, visible breast cancer lung metastasis was not observed in our experimental setting with this xenograft model. I) Primary tumor weight was measured, data are means ± SD, P<0.05 vs. control. J) Tumor tissue nuclei were used for S1P and DH-S1P measurements using LC-MS/MS methods. K) Equal amounts of tumor tissue nuclear proteins were used for Western blot analyses with the antibodies to H3-K9ac, HIF-1α, and histone H3, as indicated. Histone H3 is used as a loading control for nuclear proteins. L-N) Tumor tissues treated with vehicle or K-145 were subjected to quantitative real-time PCR for the HIF-2α, VEGFA, and SERPINE1 gene levels normalized to GAPDH. Data are means ± SD. P<0.05 vs. vehicle.

    Article Snippet: Pulldowns with S1P affinity matrices Control or S1P-coated agarose beads (Echelon Biosciences, Salt Lake City, UT, USA) equilibrated with binding buffer containing 10 mM HEPES (pH 7.8), 150 mM NaCl, 0.5% NP-40 and 1:500 protease inhibitor cocktail were mixed with nuclear extracts preincubated with or without 10 µM S1P for 25 min and rocked overnight at 4 °C ( 17 , 27 , 28 ).

    Techniques: Inhibition, In Vivo, Microscopy, Staining, Western Blot, Real-time Polymerase Chain Reaction, Control, Liquid Chromatography with Mass Spectroscopy

    SphK2 enhances HIF-1α binding to the VEGF promoter. MCF-7 cells (A, B) or MDA-MB-231 cells (C) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or normoxia (white bar) for 40 min. For some experiments, MCF-7 cells transfected with vector (light gray bar) or V5-SphK2 (dark gray bar) were incubated at normoxia (C). Cells were subjected to ChIP analyses with antibodies to HIF-1α or normal IgG. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the VEGFA gene (A, C). For some experiments, ChIP-qPCR products were analyzed on a 2% Agarose gel (B). Relative binding to the VEGFA promoter is expressed as the fold enriched of input. Data are means ± SD. P < 0.05, relative to siControl (A) or vector transfectants (C), by Student’s t-test. D-H) MDA-MB-231 cells transfected with siControl or siSphK2 were treated with hypoxia (black bar) or normoxia (white bar) for 40 min and subjected to qPCR analyses with the specific primers to human SERPINE (D), OCT4 (E), ALKBH5 (F), CD44 (G), and VEGFA (H). mRNA levels of the genes, as indicated, were determined by quantitative real-time PCR and normalized to GAPDH. Data are means ± SD, P<0.05 vs. siControl, by Student’s t-test. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα-direct target gene expression by hypoxia.

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    Article Title: Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate

    doi: 10.1096/fj.201901734RR

    Figure Lengend Snippet: SphK2 enhances HIF-1α binding to the VEGF promoter. MCF-7 cells (A, B) or MDA-MB-231 cells (C) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or normoxia (white bar) for 40 min. For some experiments, MCF-7 cells transfected with vector (light gray bar) or V5-SphK2 (dark gray bar) were incubated at normoxia (C). Cells were subjected to ChIP analyses with antibodies to HIF-1α or normal IgG. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the VEGFA gene (A, C). For some experiments, ChIP-qPCR products were analyzed on a 2% Agarose gel (B). Relative binding to the VEGFA promoter is expressed as the fold enriched of input. Data are means ± SD. P < 0.05, relative to siControl (A) or vector transfectants (C), by Student’s t-test. D-H) MDA-MB-231 cells transfected with siControl or siSphK2 were treated with hypoxia (black bar) or normoxia (white bar) for 40 min and subjected to qPCR analyses with the specific primers to human SERPINE (D), OCT4 (E), ALKBH5 (F), CD44 (G), and VEGFA (H). mRNA levels of the genes, as indicated, were determined by quantitative real-time PCR and normalized to GAPDH. Data are means ± SD, P<0.05 vs. siControl, by Student’s t-test. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα-direct target gene expression by hypoxia.

    Article Snippet: Pulldowns with S1P affinity matrices Control or S1P-coated agarose beads (Echelon Biosciences, Salt Lake City, UT, USA) equilibrated with binding buffer containing 10 mM HEPES (pH 7.8), 150 mM NaCl, 0.5% NP-40 and 1:500 protease inhibitor cocktail were mixed with nuclear extracts preincubated with or without 10 µM S1P for 25 min and rocked overnight at 4 °C ( 17 , 27 , 28 ).

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Targeted Gene Expression

    SphK2 enhances HIF-1α protein binding to the HIF-2α promoter. MDA-MB-231 cells (A, B) or MCF7 cells (E, F) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or left at normoxia (white bar) for 40 min and subjected to ChIP analyses with antibodies to HIF-1α or normal rabbit IgG, as indicated. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the HIF-2α gene. ChIP-qPCR products for HIF-2α were analyzed on a 2% Agarose gel (B, F). MDA-MB-231 cells (C, D) or MCF-7 cells (G, H) were transfected with vector (light gray) or V5-SphK2 (dark gray) and processed for ChIP analyses with antibodies to HIF-1α or normal control IgG. ChIP-PCR products were analyzed on a 2% Agarose gel, and representative images are shown (D, H). Relative binding to the promoter using ChIP-qPCR data is expressed as the fold enriched of input. Data are means ± SD, P<0.05, relative to siControl (A, E), or vector transfectants (C, G), respectively. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα positive feedback regulation by hypoxia.

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    Article Title: Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate

    doi: 10.1096/fj.201901734RR

    Figure Lengend Snippet: SphK2 enhances HIF-1α protein binding to the HIF-2α promoter. MDA-MB-231 cells (A, B) or MCF7 cells (E, F) transfected with siControl or siSphK2 were exposed to hypoxia (black bar) or left at normoxia (white bar) for 40 min and subjected to ChIP analyses with antibodies to HIF-1α or normal rabbit IgG, as indicated. The precipitated DNA was analyzed by real-time PCR with primers amplifying the core promoter sequence of the HIF-2α gene. ChIP-qPCR products for HIF-2α were analyzed on a 2% Agarose gel (B, F). MDA-MB-231 cells (C, D) or MCF-7 cells (G, H) were transfected with vector (light gray) or V5-SphK2 (dark gray) and processed for ChIP analyses with antibodies to HIF-1α or normal control IgG. ChIP-PCR products were analyzed on a 2% Agarose gel, and representative images are shown (D, H). Relative binding to the promoter using ChIP-qPCR data is expressed as the fold enriched of input. Data are means ± SD, P<0.05, relative to siControl (A, E), or vector transfectants (C, G), respectively. I) A model is shown demonstrating the epigenetic role of nuclear S1P-mediated HIFα positive feedback regulation by hypoxia.

    Article Snippet: Pulldowns with S1P affinity matrices Control or S1P-coated agarose beads (Echelon Biosciences, Salt Lake City, UT, USA) equilibrated with binding buffer containing 10 mM HEPES (pH 7.8), 150 mM NaCl, 0.5% NP-40 and 1:500 protease inhibitor cocktail were mixed with nuclear extracts preincubated with or without 10 µM S1P for 25 min and rocked overnight at 4 °C ( 17 , 27 , 28 ).

    Techniques: Protein Binding, Transfection, Real-time Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation, Control, Binding Assay

    Nuclear S1P directly binds to HIFα and promotes activation in response to hypoxia.

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    Article Title: Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate

    doi: 10.1096/fj.201901734RR

    Figure Lengend Snippet: Nuclear S1P directly binds to HIFα and promotes activation in response to hypoxia.

    Article Snippet: Pulldowns with S1P affinity matrices Control or S1P-coated agarose beads (Echelon Biosciences, Salt Lake City, UT, USA) equilibrated with binding buffer containing 10 mM HEPES (pH 7.8), 150 mM NaCl, 0.5% NP-40 and 1:500 protease inhibitor cocktail were mixed with nuclear extracts preincubated with or without 10 µM S1P for 25 min and rocked overnight at 4 °C ( 17 , 27 , 28 ).

    Techniques: Activation Assay

    Sphk1-mediated production of S1P functions intracellularly to regulate GAPDH and Hb localization and subsequent metabolic consequences. ( a , b ) Total and membrane bound GAPDH protein levels in WT, Sphk1 −/− , SCD and SCD/Sphk1 −/− mouse erythrocytes detected by western blot (cropped blots displayed, whole blots see supplementary information). Cytosolic GAPDH activity ( c ) and membrane bound heme ( d ) in WT, Sphk1 −/− , SCD and SCD/Sphk1 −/− mouse erythrocytes. Cytosolic GAPDH activity ( e ), membrane bound heme ( f ), 2,3-BPG levels ( g ), NADPH levels ( h ) and percentage of sickled erythrocytes in SCD/Sphk1 −/− mouse erythrocytes treated with different concentration of S1P. Values shown represent the mean ± SEM (n = 5); * p < 0.05 versus WT or 2.5 µM; ** p < 0.05 versus SCD or 5 µM, Student’s t -test.

    Journal: Scientific Reports

    Article Title: Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease

    doi: 10.1038/s41598-017-13667-8

    Figure Lengend Snippet: Sphk1-mediated production of S1P functions intracellularly to regulate GAPDH and Hb localization and subsequent metabolic consequences. ( a , b ) Total and membrane bound GAPDH protein levels in WT, Sphk1 −/− , SCD and SCD/Sphk1 −/− mouse erythrocytes detected by western blot (cropped blots displayed, whole blots see supplementary information). Cytosolic GAPDH activity ( c ) and membrane bound heme ( d ) in WT, Sphk1 −/− , SCD and SCD/Sphk1 −/− mouse erythrocytes. Cytosolic GAPDH activity ( e ), membrane bound heme ( f ), 2,3-BPG levels ( g ), NADPH levels ( h ) and percentage of sickled erythrocytes in SCD/Sphk1 −/− mouse erythrocytes treated with different concentration of S1P. Values shown represent the mean ± SEM (n = 5); * p < 0.05 versus WT or 2.5 µM; ** p < 0.05 versus SCD or 5 µM, Student’s t -test.

    Article Snippet: Approximately 100 µl of various lipids conjugated to agarose beads including S1P-agarose beads, lysophosphatic acid-beads or sphingosine-beads (Echelon Biosciences Inc, Salt Lake City, UT) were washed twice with lysis buffer.

    Techniques: Western Blot, Activity Assay, Concentration Assay

    Functional and structural evidence for S1P binding to Hb and stabilization of deoxyHb in T-state. ( a ) Pull-down of Hb by lysophosphatidic acid (LPA), Sphingosine and S1P beads from normal human and SCD patient RBC lysates (cropped blots displayed, for full blots see supplementary information). S1P, at physiological and pathological molar ratios, induces further O 2 release from HbA ( b ) and HbS ( c ) in the presence of 2,3-BPG. ( d ) Crystal structure of deoxyHbA in complex with 2,3-BPG (bound at the β-cleft), and S1P (bound both at the central water cavity and the protein surface). ( e ) Close view of 2,3-BPG binding at the β-cleft. ( f – h ) S1P binds to the surface of HbA in the presence of 2,3-BPG and induces further conformational change stabilizing the complex in T-state. ( i ) Working model: elevated erythrocyte Sphk1 activity increases production of S1P, which binds to deoxyHbS and facilitates deoxyHbS anchoring to membrane and release of GAPDH. Increased cytosolic GAPDH accelerates glycolysis and 2,3-BPG production while decreasing PPP and antioxidant production. Increased 2,3-BPG leads to more deoxyHbS and more sickling while decreased antioxidant causes more oxidative stress (ROS) and more hemolysis. Altogether, erythrocyte S1P induced by elevated Sphk1 activity leads to impaired metabolic reprogramming and thus underlies sickling, hemolysis and disease progression in SCD.

    Journal: Scientific Reports

    Article Title: Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease

    doi: 10.1038/s41598-017-13667-8

    Figure Lengend Snippet: Functional and structural evidence for S1P binding to Hb and stabilization of deoxyHb in T-state. ( a ) Pull-down of Hb by lysophosphatidic acid (LPA), Sphingosine and S1P beads from normal human and SCD patient RBC lysates (cropped blots displayed, for full blots see supplementary information). S1P, at physiological and pathological molar ratios, induces further O 2 release from HbA ( b ) and HbS ( c ) in the presence of 2,3-BPG. ( d ) Crystal structure of deoxyHbA in complex with 2,3-BPG (bound at the β-cleft), and S1P (bound both at the central water cavity and the protein surface). ( e ) Close view of 2,3-BPG binding at the β-cleft. ( f – h ) S1P binds to the surface of HbA in the presence of 2,3-BPG and induces further conformational change stabilizing the complex in T-state. ( i ) Working model: elevated erythrocyte Sphk1 activity increases production of S1P, which binds to deoxyHbS and facilitates deoxyHbS anchoring to membrane and release of GAPDH. Increased cytosolic GAPDH accelerates glycolysis and 2,3-BPG production while decreasing PPP and antioxidant production. Increased 2,3-BPG leads to more deoxyHbS and more sickling while decreased antioxidant causes more oxidative stress (ROS) and more hemolysis. Altogether, erythrocyte S1P induced by elevated Sphk1 activity leads to impaired metabolic reprogramming and thus underlies sickling, hemolysis and disease progression in SCD.

    Article Snippet: Approximately 100 µl of various lipids conjugated to agarose beads including S1P-agarose beads, lysophosphatic acid-beads or sphingosine-beads (Echelon Biosciences Inc, Salt Lake City, UT) were washed twice with lysis buffer.

    Techniques: Functional Assay, Binding Assay, Activity Assay

    Crystallographic data for  deoxyHbA-S1P-2,3-BPG  and deoxyHbA-S1P complex structures.

    Journal: Scientific Reports

    Article Title: Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease

    doi: 10.1038/s41598-017-13667-8

    Figure Lengend Snippet: Crystallographic data for deoxyHbA-S1P-2,3-BPG and deoxyHbA-S1P complex structures.

    Article Snippet: Approximately 100 µl of various lipids conjugated to agarose beads including S1P-agarose beads, lysophosphatic acid-beads or sphingosine-beads (Echelon Biosciences Inc, Salt Lake City, UT) were washed twice with lysis buffer.

    Techniques: