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Salas, A. et al. Sphingosine kinase-1 and sphingosine 1-phosphate receptor 2 mediate Bcr-Abl1 stability and drug resistance by modulation of protein phosphatase 2A. Blood 117, 5941–5952. https://doi.org/10.1182/blood-2010-08-300772 (2011). Ogretmen, B. Sphingolipid metabolism in cancer signalling and therapy. Nat. Rev. Cancer 18, 33–50. https://doi.org/10.1038/nrc.2017.96 (2018). Purified Des1-FLAG protein was assayed with NBD-C6-dhCer in 1% fatty acid-free BSA, 1 mM NADPH, 50 µM (NH 4) 2Fe(SO 4) 2 (prepared with 3 × molar excess ascorbic acid) and recombinant cytochrome B5 (CYB5) in 50 mM Tris–HCl buffer (pH 7.2) with 1 mM DTT. The reaction was incubated at 37 °C for 30 min, and then terminated by the addition of methanol and centrifugation at 17,000× g for 5 min. Lipid extracts were transferred to glass HPLC vials and analysed as above. S1P lyase assay

Size references have been modified to match BS1806 and AS568, however there are two tolerance standards in existance that do not necessarily match are usually free or discounted: Lawyer Referral & Information Service (LRIS) Committed to Public Service Green, C. D., Maceyka, M., Cowart, L. A. & Spiegel, S. Sphingolipids in metabolic disease: The good, the bad, and the unknown. Cell Metab. 33, 1293–1306. https://doi.org/10.1016/j.cmet.2021.06.006 (2021). Osada, M., Yatomi, Y., Ohmori, T., Ikeda, H. & Ozaki, Y. Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist. Biochem. Biophys. Res. Commun. 299, 483–487. https://doi.org/10.1016/s0006-291x(02)02671-2 (2002). 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.Molecular Therapeutics Laboratory, Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, Australia Bonhoure, E. et al. Overcoming MDR-associated chemoresistance in HL-60 acute myeloid leukemia cells by targeting sphingosine kinase-1. Leukemia 20, 95–102. https://doi.org/10.1038/sj.leu.2404023 (2006). Pitman, M.R., Lewis, A.C., Davies, L.T. et al. The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia Pitman, M. R., Pham, D. H. & Pitson, S. M. Isoform-selective assays for sphingosine kinase activity. Methods Mol. Biol. 874, 21–31. https://doi.org/10.1007/978-1-61779-800-9_2 (2012). 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 Drexler, Y., Molina, J., Mitrofanova, A., Fornoni, A. & Merscher, S. Sphingosine-1-phosphate metabolism and signaling in kidney diseases. J. Am. Soc. Nephrol. 32, 9–31. https://doi.org/10.1681/ASN.2020050697 (2021).

French, K. J. et al. Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res. 63, 5962–5969 (2003).

Custodia, A. et al. Ceramide metabolism and Parkinson’s disease-therapeutic targets. Biomolecules https://doi.org/10.3390/biom11070945 (2021). 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).

Park, S. J. & Im, D. S. Deficiency of sphingosine-1-phosphate receptor 2 (S1P2) attenuates bleomycin-induced pulmonary fibrosis. Biomol. Ther. 27, 318–326. https://doi.org/10.4062/biomolther.2018.131 (2019). The relationship between sphingosine-1-phosphate receptor 2 and epidermal growth factor in migration and invasion of oral squamous cell carcinoma Fraud is a constant threat, and those who commit fraud take money away from public services and those who rely on them. The increase in fraud threat during COVID-19 reminds us that fraud is an increasingly complex and dynamic crime, and it requires skill, capability and commitment to mitigate it effectively. 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).

Saba, J. D. Fifty years of lyase and a moment of truth: Sphingosine phosphate lyase from discovery to disease. J. Lipid. Res. 60, 456–463. https://doi.org/10.1194/jlr.S091181 (2019). Using an internal standard approach, 58 sphingolipid species were identified based on accurate mass and retention time, with the majority of sphingolipids detected with excellent precision (RSD < 10%). Quantitation was performed by semi-automated peak integration using Tracefinder 3.2 (Thermo Fisher) with manual verification. The sample concentrations were calculated based on the ratio of peak area of each identified lipid component over the area of the corresponding internal standard (C17 ceramide was used as internal standard for all ceramides). Concentrations were then converted to pmol/1 × 10 6 cells by dividing calculated concentration by cell number. For simplicity of nomenclature, ceramides with no double bonds were defined as dihydroceramides, those with one double bond defined as ceramides, and those with two double bonds defined as ceramides with unsaturated N-linked acyl chains (noting that the latter two classes may contain small contributions from isomeric dihydroceramides with unsaturated N-linked acyl chains). Sphingosine kinase assaysKang, 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). The publication of the Counter Fraud Functional Standard reinforces the government’s commitment to fighting fraud. By meeting the requirements of this standard, public bodies are better prepared, and more able to respond to the risk of fraud against the public sector. Human S1P lyase cDNA (SGPL1, Genbank Accession number NM_003901) was amplified from placenta cDNA and FLAG epitope-tagged at the 3′ end by PCR with oligonucleotide primers 5′-TATATAGAATTCGCCACCATGCCTAGCACAGACCTTCT-3′ and 5′-TATATAGAATTCACTTGTCATCGTCGTCCTTGTAGTCGTGGGGTTTTGGAGAACCAT-3′. The PCR product was digested with EcoRI and cloned into pcDNA3 (Invitrogen) for expression in mammalian cells. Sequencing verified the orientation and integrity of the cDNA. Quantitative RT-PCR SK1 degradation assays were performed as described previously 47. JTE-013 treatments were compared to DMSO vehicle or SKi positive control in the presence or absence of proteasome inhibitor MG132 (10 µM). Cell survival assays Li, C. et al. Sphingosine 1-phosphate receptor 2 antagonist JTE-013 increases the excitability of sensory neurons independently of the receptor. J. Neurophysiol. 108, 1473–1483. https://doi.org/10.1152/jn.00825.2011 (2012).