3 3h d erythro sphingosine

Sphingosine kinase activity was performed according to the standard protocol as described elsewhere [62 (link)]. Briefly, Sphingosine kinase activity was measured from cell lysates in the presence of 20 μmol/L sphingosine, 1 mmol/L ATP, 10 mmol/L MgCl2, and 10 μCi/mL [3-³H] D-erythro-sphingosine (PerkinElmer, San Diego, USA). Sphk1 activity was determined in the presence of 0.25% Triton X-100, which inhibits Sphk2 activity. Sphk2 activity was determined with sphingosine added as a complex with 4 mg/mL BSA and 1 mol/L KCl, conditions in which Sphk2 activity is optimal and activity of Sphk1 is strongly inhibited. The reaction was performed at 37°C for 30 min and stopped on ice.

All chemical reagents were of analytical grade or higher. Lipid standards where from Avanti Polar Lipids (Alabaster, USA) or Matreya LLC (Pleasant Gap, USA). Cholesteryl phosphocholine (CholPC) was synthesized as described by Lönnfors et al. [38 (link)], or obtained from Avanti Polar Lipids. D-erythro-sphingosine and hexanoic acid were purchased from Larodan (Malmö, Sweden). [3-³H]D-erythro-sphingosine (15–30 Ci/mmol) and [9–10,³H]hexadecanoic acid (30–60 Ci/mmol) were obtained from PerkinElmer (Waltham, MA, USA). Decanoic and hexadecanoic acids were obtained from Sigma-Aldrich (St. Louis, MO, USA). Organic solvents were from Rathburn Chemicals Ltd (Walkerburn Scotland). HeLa cells (ATCC CCL-2, LGC Standards) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with penicillin (50 U/ml), streptomycin (50 U/ml), 4 mM L-glutamine and 10% fetal calf serum (FCS, all from Sigma-Aldrich) prior to the experiments. Chloroquine and ceranib-2 were purchased from Sigma-Aldrich and fumonisin B1 was purchased from Enzo Life Sciences (Farmingdale, NY, USA).