L 3h g serine

Pyridoxal-5’-phosphate, L-Serine, and palmitoyl coenzyme A were purchased from Sigma-Aldrich (St. Louis, MI, USA). Sphingosine-1-phosphate (S1P) was purchased from Avanti Polar Lipids. DMEM culture media and fetal bovine serum (FBS) were purchased from EuroClone Life Science Division (Milan, Italy). l-[³H(G)]-Serine was purchased from PerkinElmer, Inc. (Wellesley, MA, USA).
Human breast cancer cell line MDA-MB-231, from America Type Culture Collection (ATCC), Rockville, MD, USA, was chosen as the source of serine-palmitoyl transferase (SPT) enzyme. Cells were maintained at 5% CO₂, 95% humidity at 37 °C in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin solution as antibiotics. Cells were seeded in 150 mm Petri dishes (3 × 106 cells in 24 mL) and left to grow for at least 3 days, until 80–90% confluence was reached, before being collected for microsomal membranes fraction extraction, due to the localization of the studied enzyme at this level.

Cells were precultured overnight in EMM + E or EMM + S medium. 2 × 10ˆ6 cells were suspended in 400 μL of EMM + E or EMM + S medium. 100 μL of medium containing 2 μCi of [¹⁴C]-glutamate or [³H]-serine was added to the cell suspension, incubated at 27°C for 0.5, 1, or 2 h. Reaction was quenched by ice-cold medium containing 10 mM glutamate or serine (500 μL). Cells were washed by 500 μL of ice-cold medium three times and suspended in 0.5% SDS (100 μL), which was mixed with Scintillation Cocktail (2 mL, Ultima Gold from Perkin Elmer). The RI count was measured using Tri-Carb 4810TR (Perkin Elmer). l-[¹⁴C(U)]-glutamic acid (281.0 mCi/mmol, 50 μCi/0.5 mL) and l-[³H(G)]-serine (30.5 mCi/mmol, 250 μCi/0.25 mL) were purchased from Perkin Elmer.