<i>N. meningitidis</i> infection activates ASM and causes membrane ceramide accumulation on HBMEC.

<p>(A) ASM activity was measured in whole cell lysates of HBMEC infected with <i>N. meningitidis</i> wildtype strain MC58 or not (control) after the indicated time intervals. Data (mean ± S.D.) were generated in three independent experiments, each performed in triplicate. (B to E) Surface ASM and ceramide levels on infected HBMEC were determined by flow cytometry analysis or spot assays (shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004160#ppat-1004160-g002" target="_blank">Fig. 2D</a>, insert). (B) Surface display of ASM (upper row, purple line) and ceramide (bottom row, red line) on HBMEC infected with <i>N. meningitidis</i> strain MC58 for 4 hrs, control, uninfected HBMEC. An isotype antibody served as a negative control in staining experiments (both rows, green line) (MFI of isotype control ceramide = 9.79±1.34; MFI of isotype control ASM = 13.12±0.53). (C) Surface ASM levels on infected HBMEC were determined in three independent experiments performed in duplicates. (D) Surface ceramide release on wildtype strain MC58 infected HBMEC (and, for control, uninfected cells) was determined by flow cytometry or spot assays (inset). An isotype antibody served as a negative control in staining experiments. For the spot assay, HBMEC were infected with wildtype strain MC58 and an isogenic, unencapsulated isolate MC58 <i>siaD</i> or were left uninfected (control) for 2 hrs. (E) Surface ceramide levels were estimated on HBMEC treated with amitriptyline (open circles) or not (filled circles) prior to infection (MFI of isotype control ceramide = 8.63±0.94). (F) Detection of ASM or ceramides on HBMEC infected with MC58 (lower panel) or left uninfected (control, upper panel) by microscopy. As representatively shown, ASM co-localized in ceramide-enriched membrane platforms on HBMEC upon infection with <i>N. meningitidis</i>. Size bars represent 10 µm. All data show mean values ± S.D. of three independent experiments done in duplicate. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, relative to uninfected control cells or to cells infected without inhibitor. Fluorescence microscopy studies are representative for 3 similar studies.