Fig. 2: Trifunctional sphingomyelins (TFSMs) are taken up and metabolized by human cells.
a Sphingomyelinase Förster resonance energy transfer (FRET) probe is cleaved by recombinant human acid sphingomyelinase (rhASM) but not bacterial sphingomyelinase (bSMase). rhASM or bSMase were incubated in the presence of FRET probe and probe conversion was monitored by measurement of FITC fluorescence in a microplate reader. n = 3. b, c TFSMs are incorporated into human cells. HEK293T cells were treated with TFSM 1 or TFSM 2 for 24 h at 37 °C. After cell lysis, amounts of TFSMs, ceramide (Cer), and sphingosine (Sph) were detected by LC-MS/MS. Internal standards were used for each metabolite type, and their peak areas were used to normalize the peak area of the corresponding TFSM metabolite. n = 3. d Cellular uptake of TFSMs is affected by fetal bovine serum (FBS). HeLa cells were treated with TFSM 1 in the presence of 1 or 10% FBS for 2 h. Samples were fixed and clicked with AlexaFluorTM (AF)488-DBCO (backbone) or Cy5-azide (headgroup). n = 1. Scale bars: 50 µm. e Glutaraldehyde (GA) fixation of TFSMs. HeLa cells were incubated with BODIPY-FL-C12-sphingomyelin (BODIPY-FL-C12-SM), TFSM 1 or TFSM 2 for 2 h, bSMase-treated, detached, fixed, and permeabilized with TX-100 (as indicated). Fixation was done either with paraformaldehyde (PFA) or PFA and GA. TFSM then were BODIPY-FL-DBCO-stained and analyzed by flow cytometry. n = 3 (BODIPY-FL-C12-SM, TFSM 1 PFA and TFSM 2 PFA), n = 6 (TFSM 1 GA + PFA, TFSM 2 GA + PFA). Statistics: Two-way ANOVA and Šídák’s (b) or Tukey’s (e) multiple comparisons. Two-sided unpaired Student’s t test (c). Bars represent means ± SD. n corresponds to biological replicates. Source data and detailed statistics are provided as a Source Data file.
