3E2 antibody inhibits <i>ex vivo</i> angiogenesis.

<p>A) Viability measured by MTT of HMEC-1 treated for 24 h with increasing concentration of 3E2 (▾) or isotypic control (▪) (n = 3; mean±SEM; *p<0.05). B) Western blot of phosphor-ERK and phosphor-AKT from cells treated with either 40 µg/ml 3E2 or IgM-control antibody (n = 3). C) Photographs of sprouting vessels from aorta 5 d post-treatment by an increasing dose of 3E2 (n = 3). D) Sprouting index from aorta rings 5 d post-treatment with 3E2 or isotypic control (n = 3, mean±SEM; *p<0.05).</p

3E2 targets Gb3 on the endothelial cell membrane.

<p>A) Gb3 immunolocalization in HMEC-1 by FITC-conjugated 3E2 (green; upper panel) <i>vs.</i> control isotypic 11E10 IgM (lower panel) and counterstaining with Draq5. Scale bar represent 20 µm. B) Percentage of Gb3-positive cells by Facs analysis after hybridization of HMEC-1, RAJI and NXS2 cells with using 3E2 or isotypic control. C) GSLs profiles by orcinol staining after HPTLC (panel a) and Gb3 expression by immunohybridization using 3E2 (panel b) or commercially available 38.13 (panel c). Lane 1: rat brain gangliosides; lane 2: neutral GSLs mixture: lane 3, purified Gb3; lane 4: HMEC-1 glycolipids extract.</p

3E2 inhibits in <i>vivo</i> metastases spreading.

<p>A) Pictures of NXS2 hepatic metastases by confocal microscopy stained with Alexa488-conjuguated CD31 mAb (green), Alexa568-conjuguated 3E2 (red) or isotypic IgM control and counterstained with Draq5. Colocalization of the endothelial marker CD31 and Gb3 is shown by the yellow staining on the merge image. B) Number of liver metastases per animal (n = 6; mean±SEM; *p<0.05). C) Representative photography of liver 28 days after NXS2 injection and the different immunotherapies (scale bar represents 1 cm).</p

3E2 antibody inhibits endothelial cell proliferation.

<p>HMEC-1 cells are tracked by videomicroscopy up to 24 h after treatment by 3E2 or isotypic control. A) Cell number per field in function of time. Histograms show the mean doubling time (n = 6 for IgM and n = 9 for 3E2, mean±SEM; *p<0.05). B) Microphotographs of representative fields of HMEC-1 in function of time. Magnification 10×. C) Number of mitosis summed every 6 h for 24 h (n = 5, mean±SEM; *p<0.05). D) Duration of the mitosis (n = 5, mean±SEM; *p<0.05). E) Cell death quantification detected by sub-G1 and hoechst assays from 3E2- or IgM-treated HMEC-1 (n = 3; mean±SD; ns: p>0.1).</p

3E2 mitigates tumor growth.

<p>A) Tumor volume of NXS2 tumors (n = 7; mean±SEM; *p<0.05). B) Doubling time of the each tumors (•) estimated during the four first days of the different treatments (bars: mean±SEM; *p<0.01; n = 7). C) Actuarial Morbidity curves of to tumor reaching 300 mm³ (n = 7). D) Vascular density estimated on 50 different slides on 3 different treated tumors (mean±SEM; *p<0.01; n = 3). E) Mosaic pictures by apotome microscopy stained with Alexa488-conjuguated CD31 mAb (green) and counterstained with Draq5 (bar: 500 µm).</p

Distribution of Gb3 in organs from C57Bl/6 mouse obtained after staining with Alexa568-conjuguated 3E2.

<p>Frozen healthy organs sections of 5 µm were hybridized with biotinylated 3E2 or its isotypic control, revealed by an Alexa 568-conjugated streptavidin and counterstained with DAPI. Pictures were observed under a confocal microscope (n = 3).</p>#<p>: High background.</p>*<p>: Lipofuscin autofluorescence.</p

Gb3 is over-expressed in proliferating endothelial cells.

<p>HMEC-1 were treated with medium containing respectively 15% serum, 0.1% serum and 0.1% serum with 1 µM S1P or its vehicle. A) Proliferation assay by ³H-thymidine incorporation 24 h after treatment. (n = 3; mean±SD; *p≤0.05). B) RT-Q PCR 24 h after treatment (n = 6; mean±SEM; ns: p<0.01). C) Gb3 expression detected by 3E2 after immuno-HPLC. D) Gb3-positive HMEC-1 determines by Facs using 10 µg/ml of 3E2 (n = 3). E). Gb3 site number determined by Scatchard analysis using 3E2.</p

Gb3 is not modulated by 3E2 antibody treatment.

<p>HMEC-1 cells were treated with either 40 µg/ml 3E2- or IgM control-antibody. A) RT-Q PCR 24 h after treatment (n = 6; mean±SEM; ns: p>0.1). B) Gb3-positive HMEC-1 cells determined by Facs 24 h after treatment (n = 3). C) Table analysis (% of positive cells, means) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045423#pone-0045423-g003" target="_blank">Fig. 3 B</a> (ns>0.1).</p