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M.R.P., A.C.L., L.T.D., P.A.B.M., D.A., D.J.C. and J.A.P. performed experiments. M.R.P., A.C.L. and S.M.P. drafted the manuscript, and all authors reviewed the manuscript. Corresponding authors McNaughton, M., Pitman, M., Pitson, S. M., Pyne, N. J. & Pyne, S. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells. Oncotarget 7, 16663–16675. https://doi.org/10.18632/oncotarget.7693 (2016). Long, J. S. et al. Sphingosine 1-phosphate receptor 4 uses HER2 (ERBB2) to regulate extracellular signal regulated kinase-1/2 in MDA-MB-453 breast cancer cells. J. Biol. Chem. 285, 35957–35966. https://doi.org/10.1074/jbc.M110.117945 (2010). Salomone, S. & Waeber, C. Selectivity and specificity of sphingosine-1-phosphate receptor ligands: Caveats and critical thinking in characterizing receptor-mediated effects. Front. Pharmacol. 2, 9. https://doi.org/10.3389/fphar.2011.00009 (2011). Venant, H. et al. The sphingosine kinase 2 inhibitor ABC294640 reduces the growth of prostate cancer cells and results in accumulation of dihydroceramides in vitro and in vivo. Mol. Cancer Ther. 14, 2744–2752. https://doi.org/10.1158/1535-7163.MCT-15-0279 (2015).

Price, S. T. et al. Sphingosine 1-phosphate receptor 2 regulates the migration, proliferation, and differentiation of mesenchymal stem cells. Int. J. Stem Cell Res. Ther. 2, 014. https://doi.org/10.23937/2469-570x/1410014 (2015). Kang, J., Lee, J. H. & Im, D. S. Topical application of S1P2 antagonist JTE-013 attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice. Biomol. Ther. 28, 537–541. https://doi.org/10.4062/biomolther.2020.036 (2020).

Lim, K. G. et al. Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: Functional differences between sphingosine kinase 1a and 1b. Int. J. Biochem. Cell Biol. 44, 1457–1464. https://doi.org/10.1016/j.biocel.2012.05.012 (2012). French, K. J. et al. Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res. 63, 5962–5969 (2003). Brinkmann, V. et al. Fingolimod (FTY720): Discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897. https://doi.org/10.1038/nrd3248 (2010).

MV411 cells (1.5 × 10 7) were seeded at 1 × 10 6 per mL and treated with DMSO (0.1% final) or 10 µM JTE-013 for 6 h. Cells were pelleted by centrifugation, washed in PBS and snap frozen. The cell pellets were suspended in 1 mL of chilled PBS and centrifuged at 2000× g for 5 min at 4 °C. The supernatant was discarded and the remaining solid was resuspended in 450 µL of extraction mix [chloroform: methanol: H 2O, 2:6:1 (v/v)]. Odd chain lipid standards mix was added to give final concentrations of 241 nM sphingosine (d17:1), 250 nM dihydosphingosine (d17:0), 253 nM S1P (d17:1), 235 nM dihydrosphingosine 1-phosphate (d17:0), 250 nM sphingomyelin (d18:1/17:0), and 450 nM C17 ceramide (d18:1/17:0). The samples were frozen/thawed three times, then centrifuged at 14,800× g for 5 min at 4 °C. 400 µL of supernatant was then transferred to new tubes, with some remaining sample combined to make a pooled QC sample. To reconstitute, the solvent was evaporated to dryness keeping the samples in a centrifugal evaporator at 55 °C for 50 min. Dried extracts were frozen at – 80 °C until LC–MS analysis was performed. On the day of analysis, the samples were dissolved in 180 µL of butanol: methanol (v/v 1:1) mixture and 20 µL of water. The samples were vortexed on a rotary vortex for 10 min and sonicated in a sonicator bath for 1 h keeping the temperature below 25 °C. The samples were centrifuged at 14,800× g for 10 min at 20 °C and transferred to LC–MS vials.This standard also includes groove recommendations for staic, hydraulic and pneumatic applications ISO3601 Size Ikeda, H. et al. Antiproliferative property of sphingosine 1-phosphate in rat hepatocytes involves activation of Rho via Edg-5. Gastroenterology 124, 459–469. https://doi.org/10.1053/gast.2003.50049 (2003). Powell, J. A. et al. Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1. Biochem. J. 476, 3211–3226. https://doi.org/10.1042/BCJ20190245 (2019). Analysis of HEK293T gene expression of S1PR1-5 was performed by quantitative reverse transcriptase PCR (qPCR) as previously described 13 using 5 × 10 6 HEK293T cells. Expression of the S1PR1-5 was analysed using the Rotor-Gene Q Series software (Qiagen) using the comparative quantitation method with HEK293T amplified S1PR1 used as the calibrator. Gene expression of S1PR2 in control and S1P 2 shRNA MV411 cell lines was analysed as above, normalized to GADPH and analysed using the Rotor-Gene Q Series software (Qiagen) using the comparative quantitation method with MV411 amplified S1PR2 used as the calibrator. Intact cell assay for Des1 activity Goodman, A. D., Anadani, N. & Gerwitz, L. Siponimod in the treatment of multiple sclerosis. Expert Opin. Investig. Drugs 28, 1051–1057. https://doi.org/10.1080/13543784.2019.1676725 (2019).

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