A 350

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, reference 850457C), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG, reference 840457C), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, reference 850375C), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG, reference 840475C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, reference 850725C), and cholesterol (Chol, reference 700000P) were from Avanti Polar Lipids (Alabaster, AL, USA). All experiments were performed in 20 mM HEPES, 150 mM NaCl, pH 7.4 (buffer A). ANTS (A-350, 8-aminonapthalene-1,3,6 trisulfonic acid), and DPX (X-1525, p-xylene-bis-pyridinium bromide) were purchased from Molecular Probes (Eugene, OR, USA).

The following reagents were used: THC (THC Pharm GmbH, THC-1099), pepstatin A (Enzo Life Sciences, ALX-260-085), E64d (Enzo Life Sciences, BML-PI107), myriocin (ISP-1, Sigma-Aldrich, M1177), sphingomyelinase (_EC 3.1.4.12) from Bacillus cereus (Sigma-Aldrich, S7651), o-phenanthroline (Sigma-Aldrich, 131377) and CA-074 methyl ester (CTSB/cathepsin B inhibitor; Sigma-Aldrich, C5857). Phosphatidylcholine from egg yolk (PC; Lipid Products, grade 1, 840051P), sphingomyelin, (SM; Avanti Polar Lipids, 860061), C12 ceramide (C12-Cer; Avanti Polar Lipids, 860512), C16 dihydroceramide (C16-dhCer; Avanti Polar Lipids, 860634), palmitoyl-oleoylphosphatidylcholine (POPC; Avanti Polar Lipids, 850457) and cholesterol (Ch; Avanti Polar Lipids, 700000). DEGS1 inhibitor GT11, and dihydrosphingomyelin (dhSM) were synthesized in our laboratories. dhSM was synthesized from egg SM (Avanti Polar Lipids, 860061) and contained 86% C16 dhSM. ANTS (Molecular Probes, Inc., A350), DPX (Molecular Probes, Inc., X1525) and DiIC₁₈ (Molecular Probes, D3911). BODIPY FL C5-ceramide complexed to BSA (ThermoFisher Scientific, B22650).