2(013)

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Our findings highlight the difficulty in developing selective agents that target the lipid binding pockets of sphingolipid enzymes and receptors and raises a note of caution for using JTE-013 at concentrations above 1 µM in cell-based studies for mechanistic studies. In this study we assessed the impact of JTE-013 on sphingolipid metabolism and found broad effects that appear to result from its inhibition of both Des1 and the SKs.

As expected, the SK1/SK2/Des1 inhibitor SKi caused degradation of SK1 that was reversed by co-treatment with the proteasome inhibitor MG132 (Fig. Results are presented relative to S1P 1 and are representative of triplicate measurements in two independent experiments, mean ± SEM. The sphingolipid pathway is comprised of a range of bioactive lipids that contribute to cellular membranes and can act as signaling molecules.c) Des1-FLAG was overexpressed in HEK293T cells for 24 h, purified using FLAG-agarose beads and eluted with FLAG peptide. We have previously demonstrated that SK1 inhibition induced Mcl-1 degradation and cell death in AML cells, and that this could be recapitulated by the S1P 2 antagonist JTE-013, implicating the SK1-S1P-S1P 2 axis in controlling Mcl-1 protein levels 13. While JTE-013 shows selectivity to S1P 2/4 over other S1P receptors, the specificity of this molecule has not been very well examined. Doxycycline-inducible S1P 2 shRNA and non-targeting control MV411 cell lines were treated with or without doxycycline (1 µg/ml) for 48 h prior to analysis. Survival assays were carried out as described previously 13 using annexin V-FITC (Roche) negativity exclusion using flow cytometry (Gallious, Beckman Coulter).

Only the more abundant dhCer and Cer species are shown, with quantitation of the full list of species shown in Supplemental Table S1. Treatment of HEK293T cells with CYM5520 induced a marked increase in phospho-ERK1/2 (a downstream marker of S1P 2 activation).The most substantial fold-change in sphingolipids induced by JTE-013 were dihydroceramides, suggesting the potential for additional inhibition of dihydroceramide desaturase 1 (Des1) by JTE-013. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. At 24 h post transfection, HEK293T cells were harvested, pelleted and snap frozen in liquid nitrogen.

The certificate must include the name of the course, the name of the course provider, and the completion date.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).

d) To test if S1P 2 agonism could rescue JTE-013-induced Des1 inhibition, HEK293T cells were labelled with NBD-C6-dhCer and treated with either vehicle control (DMSO, 0.

Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. a) Purified recombinant human sphingosine kinases 1 and 2 were assayed with vehicle (DMSO), or varying doses of JTE-013. The protein concentration was determined by Bradford assay (BioRad) and 30 µg of lysate was used per assay. The cell pellets were suspended in 1 mL of chilled PBS and centrifuged at 2000× g for 5 min at 4 °C.