s1p (Echelon Biosciences)


Structured Review

S1p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/s1p/product/Echelon Biosciences
Average 92 stars, based on 1 article reviews
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

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

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

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

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