Figure 3

(A) Ceramide enriched macrodomain formation and its association with SMPD1 in stimulated endothelial cells. Semi-confluent HMECs were stimulated for 20 min with serum (5%) from healthy individuals (lower panel) or from patients with sepsis (upper panel). Subsequent to fixation and blockade of unspecific binding, immuno reactivity was tested with polyclonal anti-SMPD1 antibody and monoclonal anti-ceramide antibody using identical experimental settings in all conditions. Representative images from at least six independent experiments are shown. (B) Inhibition of SMPD1 membrane immuno reactivity and (C) ceramide enriched macrodomain formation by desipramine and NB6. Semi-confluent HMECs were pretreated for 30min with SMPD1 functional inhibitors desipramine or NB6, stimulated for 20 min with serum (5%) from healthy individuals or from patients with sepsis. Subsequent to fixation and blockade of unspecific binding, immuno reactivity was proved with (B) polyclonal anti-SMPD1 antibody and (C) monoclonal anti-ceramide antibody using identical experimental settings in all conditions. At 200 × magnification, fluorescence intensity was analyzed densitometrically using Carl Zeiss Image Examiner software. The boxes indicate the interquartile range, divided by the median (central horizontal line) as analyzed in at least 12 independent experiments. Significant differences between the patient groups w and w/o inhibitors compared with these cells treated with serum from healthy controls were calculated using t-test for paired samples (**p < 0.02), the effectiveness of inhibition (desipramine or NB6) is indicated by rhombus symbol (# p < 0.05, ## p < 0.02, compared with vehicle treated sepsis control).