Figure 1. Characterisation of SMS knockout MEFs.

(a) In vitro SMS and GCS activities were measured using C₆-NBD-ceramide as the substrate as described in the Materials and Methods. Wild type (WT) and SMS double knockout (SMS DKO) tMEFs were harvested and homogenized. Lysate (100 μg) was added to the reaction solution and incubated for 1 h at 37 °C. Fluorescent lipids were extracted and quantified using a LAS-4000 fluorescent imaging system. The results are presented as the mean ± the standard deviation (SD) (n = 4). *P < 0.005. (b) SM and ceramide levels were assessed by LC-MS/MS. The value presented is the mean ± SD (n = 3). *P < 0.005. (c) SM was stained with MBP-conjugated lysenin. Then, nuclei were stained with Hoechst 33342, and the cells were observed by confocal microscopy. Scale bars, 10 μm. (d) Membrane SM was detected with mCherry-lysenin and flowcytometer. Mean fluorescence intensity (MFI) was measured by Kaluza software. The value presented is the mean ± SD (n = 3). *P < 0.005.