Phosphatidylserine

E. coli BL21(DE3) and pET-28a(+) (Novagen, China) were applied for gene expression. The recombinant plasmid pET28a(+)-PLD was constructed and preserved in our laboratory. E. coli BL21(DE3) were cultured in a Luria-Bertani medium comprising (g/L): tryptone 10, yeast extract 5 and NaCl 10, pH 7.2. Soybean lecithin(Sigma, China) and L- serine(Aladdin, China) were used as substrates for the synthesis of PS. Phosphatidylserine(Sigma, China) were purchased to be the standard. All other chemicals and reagents used were of analytical grade.

600 pmoles of 37:4 PI4P (LM1901; Avanti) was dried in LoBind 1.5 ml Eppendorf tubes along with 20 μl 0.5 mM Phosphatidylserine (Sigma-Aldrich) and 200 μl of CHCL₃ in vacuum for 20 min at 949g. After drying of lipids, 50 μl of 10 mM Tris–HCl (pH 7.4) was added to each tube and sonicated at room temperature for 3 min. Next, 50 μl of 2× kinase assay buffer containing 40 μM ATP (Roche) was added to the tubes and 10 μg equivalent of corresponding lysate was added. Immediately after this, the tubes were transferred to a shaking incubator at 30°C for 30 min for the kinase reaction to happen. The reaction was stopped by adding 125 μl of 2.4N HCl to the reaction tubes. Then 250 μl each of CHCl₃ and methanol were added. After this, the tubes were vortexed for 2 min and centrifuged at 1,000g for 5 min to allow a clean phase separation to happen. The upper phase was punctured and the lower organic phase was transferred to a fresh LoBind 1.5 ml Eppendorf. To this tube, 500 μl of Lower Phase Wash Solution (LPWS: methanol/1 M hydrochloric acid/chloroform in a ratio of 235/245/15 [vol/vol/vol]) was added. It was vortexed for 2 min and clean phase separation was obtained by spinning tubes at 1,000g for 5 min. Again, the lower organic phase was transferred to a fresh LoBind 1.5-ml Eppendorf tube.

Membrane-forming materials: Phosphatidylcholine from soybean (≥97%, Sigma-Aldrich, St. Louis, MO, USA), prepared according to a modification of the procedure of Singleton et al. [44 (link)], was used in the experiment. Phosphatidylserine (≥97%) and lipoic acid (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used as received. The molecular weights of the PC, PS, and LA were approximately 752.08, 792.07, and 206.33 g mol⁻¹, respectively.
Electrolyte solutions: The electrolyte solutions for monolayer (pure water), interfacial tension measurements (0.1 M KCl), and microelectrophoresis (0.155 M NaCl) were prepared using water purified through a Milli-Q plus water purification system (Millipore, Burlington, MA, USA) with a resistivity of 18.2 MΩ cm.

HeLa cells were seeded in a six-well plate at 150.000 cells per well in DMEM supplemented with 10% FBS. After 24 h, cells were transfected with nSMase-2-V5, LAMP1-bSMase-V5-GFP or LAMP1-bSMase^dead-V5-GFP and grown for 24 h. Next, cells were harvested in ice-cold lysis buffer (25 mM Tris pH 7.4, 0.1 mM PMSF, 1× protease inhibitor cocktail), subjected to sonication (Branson Ultrasonic Sonifier), and centrifuged at 500×g for 10 min at 4 °C to obtain a post-nuclear fraction. Aliquots equivalent to 20 µg of total protein were included in a 100 µl of reaction mixture containing 50 mM Tris pH 7.4, 10 mM MgCl₂, 0.2% Triton X-100, 10 mM DTT, 50 µM phosphatidylserine (Sigma-Aldrich, P7769) and 50 µM C₆-NBD-SM (Biotium, 60031). Reactions were incubated at 37 °C for 2 h, terminated by addition of MeOH/CHCl₃, subjected to a Bligh and Dyer lipid extraction and then analyzed by TLC as described above.

Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, D-glucose, foetal bovine serum (FBS), L-glutamine, Trypsin, DNase I, corticosterone (CORT), Poly-L-Lysine, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), fibroblast growth factor (FGF), epidermal growth factor (EGF), and dimethyl sulfoxide were purchased from Sigma. Phosphatidylcholine (PC; from egg yolk), lysoPhosphatidylcholine (LPC; from egg yolk), phosphatidylserine (PS; from bovine brain), phosphatidylethanolamine (PE; from egg yolk), phosphatidylinositol (PI; from soy bean), phosphatidylglycerol (PG; from egg yolk), phosphatidic acid (PA; from egg yolk), sphingomyelin (SM; from egg yolk) and cardiolipin (CL; from bovine heart) were obtained in their respective salts from Sigma. B-27 supplement was obtained from Thermo Fisher Scientific.