lecule 1 expression was analyzed by confocal microscopy. HGEC seeded on gelatin-coated glass coverslips were washed with PBS at pH 7.4 and fixed with 19088077 3% paraformaldehyde. Fixed cells were directly labeled with a mouse monoclonal anti-human CD31 conjugated with FITC. At the optimal antibody concentrations, no background labeling of the nucleus and mitochondria was detected. Coverslips were mounted on glass slides using Fluoromount G 3 Stx2 and SubAB action on human microvasculature doi: 10.1371/journal.pone.0070431.g002 . Immunofluorescence images were acquired with a FluoView FV1000 confocal microscope using a Plapon 60 1.42NA oil immersion objective and images were analyzed using the Olympus FV10ASW software. Neutral red cytotoxicity assay The neutral red cytotoxicity assay was adapted from previously described protocols. HGEC were plated in gelatin coated 96-well plates and grown to confluence in complete M199 medium. The cells were then washed in PBS at pH 7.4 and exposed to Stx2 or SubAB in growth-arrested conditions for 24, 48 and 72 h. For co-treatment assays, cells were incubated with Stx2 and SubAB together, using the same MedChemExpress Piceatannol concentrations and times studied in the experiments described with each toxin alone. Then, two hundred microliters of freshly diluted neutral red in M199 was added to a final concentration of 10 g/ml and cells were incubated for an additional 1 h at 37C in 5% CO2. Cells were then washed with 200l 1% CaCl2 + 1% formaldehyde and solubilized in 200 l 1% acetic acid in 50% ethanol. Absorption in each well was read in an automated plate spectrophotometer at 540nm. Results were expressed as neutral red uptake percentage, where 100% represents control cells incubated under identical conditions but without toxin treatment. The 50% cytotoxic dose corresponds to the dilution required to kill 50% of cells. To examine the effect of C-9, cells were incubated 10037488 in the presence of C-9 per 48 h before the addition of the toxins. The cells were then incubated with or without 10 ng/ml 4 Stx2 and SubAB action on human microvasculature doi: 10.1371/journal.pone.0070431.g003 of Stx2 or 3 g/ml of SubAB for additional 24 h. Finally, cell viability was established by neutral red uptake. Gb3 expression Microscopy: Gb3 expression was analyzed by confocal microscopy. HGEC were seeded on gelatin coated glass coverslips, then washed with PBS at pH 7.4 and fixed with 3% paraformaldehyde. Fixed cells were first incubated 
overnight with a rat anti-human CD77 and then with a goat IgM anti-rat conjugated with FITC, for 2 h. Coverslips were mounted on glass slides using Fluoromount G. Immunofluorescence images were acquired with a FluoView FV1000 confocal microscope using a Plapon 60 1.42NA oil immersion objective and images were analyzed using the Olympus FV10ASW software. Thin-layer chromatography: Gb3 was detected by thin-layer chromatography. HGEC were seeded in tissue culture flasks and grown at 37C in an atmosphere of 5% CO2 until the cells were nearly confluent. Adherent cell monolayers were released from the flask by trypsinization as described above, collected by centrifugation, and resuspended in PBS at pH 7.4. Cells were washed twice with PBS at pH 7.4 to deplete the serum lipids. Total HGEC glycolipids were extracted according to the method of Bligh and Dyer. Briefly, 3 ml of chloroform: methanol 2: 1 v/v was added to the cells, and incubated on ice for 15 min. Two ml of chloroform: water was added to the tube and centrifugated at 3,0