FRET measurement of TFSM conversion. HeLa or human umbilical vein endothelial cells (HuVEC) were incubated with TFSM 1, TFSM 2, or BODIPY-FL-C 12 -SM for 2 h. Then, the compounds were removed, and cells were either treated with bSMase or left untreated for 3 h. Samples treated with TFSMs were fixed, clicked with BODIPY-FL-DBCO (backbone) and AlexaFluor TM 546 (AF)546-azide (headgroup) and FRET efficiency was determined by acceptor bleaching. Cells treated with BODIPY-FL-C 12 -SM were detached, and lipids were extracted by CHCl 3 :MeOH. The proportion of unmetabolized SM was determined by thin-layer chromatography. n = 3. Statistics: Two-way ANOVA and idak’s multiple comparisons. Bars represent means SD. n corresponds to biological replicates. Source data and detailed statistics are provided as a Source Data file.
