Bandhuvula, P., Tam, Y. Y., Oskouian, B. & Saba, J. D. The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity. J. Biol. Chem. 280, 33697–33700. https://doi.org/10.1074/jbc.C500294200 (2005). 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 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).

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).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 assays Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia

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). 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. 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 carcinomaFraud 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. Melissa R. Pitman, Alexander C. Lewis, Lorena T. Davies, Paul A. B. Moretti, Jason A. Powell & Stuart M. Pitson 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).

Wang, Z. et al. Molecular basis of sphingosine kinase 1 substrate recognition and catalysis. Structure 21, 798–809. https://doi.org/10.1016/j.str.2013.02.025 (2013). 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. This standard also includes groove recommendations for staic, hydraulic and pneumatic applications ISO3601 Size Cirillo, F. et al. The antithetic role of ceramide and sphingosine-1-phosphate in cardiac dysfunction. J. Cell Physiol. 236, 4857–4873. https://doi.org/10.1002/jcp.30235 (2021). The International Standards Organisation (ISO) standard, ISO 3601 comprehensively defines both the o-ring and hardware dimensions for rod, piston, face seal grooves.For the non-adherent MV411 cells, Des1 assays were performed as previously described 24. For assays using adherent HEK293T, the cells were harvested by trypsin and counted prior to use in the assay. Briefly, intact cells (at 1 × 10 6 cells/mL) were labeled with NBD-C6-dhCer (5 µM) in serum-free media and incubated on ice for 30 min. Cells were then pelleted and washed into fresh media (with 0.5% FBS) and dispensed into 24-well plates containing inhibitor/vehicle treatments (400 µL total) and incubated for 2 h at 37 °C and 5% CO 2. The cells were then harvested and pelleted via centrifugation at 500× g and resuspended in 100 µL H 2O. The samples were sonicated for 30 s in a bath sonicator (Diagenode) followed by the addition of 900µL cold methanol. Immediately before analysis, the samples were centrifuged for 3 min at 17,000× g and transferred to glass HPLC vials (Phenomenex). Samples (50 μL) were analysed on a Waters HPLC coupled to a fluorescence detector using a 30 cm C18 reverse-phase column (Phenomonex) eluted with 1 mL/min 20% H 2O and 80% acetonitrile with 0.1% trifluoroacetic acid. NBD-labelled substrate and product were quantitated with excitation and emission wavelengths of 465 nm and 530 nm, respectively. Des1 inhibition was calculated relative to vehicle as percentage peak area under the curve. Des1 assays using cell lysates The standard was developed by counter fraud experts (including from across the public and private sectors, banking and academia) to help guide a whole of government approach. It has been extensively tested in government before its formal release, and represents the minimum that all public bodies are expected to have in place. 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). 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). The Counter Fraud Functional Standard was launched in October 2018, and is being implemented across government. It applies to all government departments and their arms-length bodies. As of February 2020, 123 public bodies had adopted the functional standard as the basis for managing the risk of fraud, bribery and corruption in the public sector.