Figure 6
Figure 6. Endosialin binds to the vascular basement membrane in vivo. (A) Cryosections of embryonic day 15 mouse brain were incubated with mouse ES-Fc overnight at 4°C. The following day, sections were stained with FITC–anti-Fc (green) and anti-CD31, followed by Alexa Fluor 555–anti–rat IgG (red). Nuclei were counterstained with DAPI (blue). Arrowheads indicate ES-Fc binding associated with the abluminal side of the endothelial cells but not with the adjacent pericytes (arrows). Scale bar indicates 50 μm. (B) Aortic ring outgrowths from wild-type 129/SvJ mice were cultured for 6 days and then stained with ES-Fc or CD44-Fc for 1 hour at 37°C. After fixation, cultures were stained with Cy3–anti-Fc (red) and FITC–isolectin B4 (green). Nuclei were counterstained with DAPI (white). Arrowheads indicate ES-Fc binding associated with the abluminal side of the endothelial cells but not with the adjacent pericytes (arrows). Scale bar indicates 50 μm. (C) Aortic ring outgrowths were incubated with ES-Fc as described in panel B. After fixation, cultures were stained with FITC–anti-Fc (green) and antifibronectin, followed by Alexa Fluor 555–anti–rabbit Ig (red). Nuclei were counterstained with DAPI (blue). Arrowhead indicates colocalization with fibronectin on the abluminal surface of the endothelial cells. Arrow indicates lack of colocalization with fibronectin associated with the basement membrane surrounding the pericytes. Scale bar indicates 25 μm. (D) Immortalized mouse skin endothelial cells (sEND) cells were cultured for 2 days on coverslips in the presence of no drug, amiloride, and/or aprotinin. Cells were incubated with ES-Fc for 1 hour at 37°C, fixed, and stained with FITC–anti-Fc (green). Nuclei were counterstained with DAPI (white). Scale bar indicates 50 μm. Representative images are shown. Data shows quantification of ES-Fc binding from 3 independent experiments ± SEM. *P < .05. (E) sEND cells were either mock transfected or transfected with control siRNA, uPAR siRNA, uPA siRNA, or uPA plus uPAR siRNAs. Forty-eight hours after transfection, cells were incubated with ES-Fc as described in panel D. Data show quantification of ES-Fc binding from 3 independent experiments ± SEM. *P < .05.

Endosialin binds to the vascular basement membrane in vivo. (A) Cryosections of embryonic day 15 mouse brain were incubated with mouse ES-Fc overnight at 4°C. The following day, sections were stained with FITC–anti-Fc (green) and anti-CD31, followed by Alexa Fluor 555–anti–rat IgG (red). Nuclei were counterstained with DAPI (blue). Arrowheads indicate ES-Fc binding associated with the abluminal side of the endothelial cells but not with the adjacent pericytes (arrows). Scale bar indicates 50 μm. (B) Aortic ring outgrowths from wild-type 129/SvJ mice were cultured for 6 days and then stained with ES-Fc or CD44-Fc for 1 hour at 37°C. After fixation, cultures were stained with Cy3–anti-Fc (red) and FITC–isolectin B4 (green). Nuclei were counterstained with DAPI (white). Arrowheads indicate ES-Fc binding associated with the abluminal side of the endothelial cells but not with the adjacent pericytes (arrows). Scale bar indicates 50 μm. (C) Aortic ring outgrowths were incubated with ES-Fc as described in panel B. After fixation, cultures were stained with FITC–anti-Fc (green) and antifibronectin, followed by Alexa Fluor 555–anti–rabbit Ig (red). Nuclei were counterstained with DAPI (blue). Arrowhead indicates colocalization with fibronectin on the abluminal surface of the endothelial cells. Arrow indicates lack of colocalization with fibronectin associated with the basement membrane surrounding the pericytes. Scale bar indicates 25 μm. (D) Immortalized mouse skin endothelial cells (sEND) cells were cultured for 2 days on coverslips in the presence of no drug, amiloride, and/or aprotinin. Cells were incubated with ES-Fc for 1 hour at 37°C, fixed, and stained with FITC–anti-Fc (green). Nuclei were counterstained with DAPI (white). Scale bar indicates 50 μm. Representative images are shown. Data shows quantification of ES-Fc binding from 3 independent experiments ± SEM. *P < .05. (E) sEND cells were either mock transfected or transfected with control siRNA, uPAR siRNA, uPA siRNA, or uPA plus uPAR siRNAs. Forty-eight hours after transfection, cells were incubated with ES-Fc as described in panel D. Data show quantification of ES-Fc binding from 3 independent experiments ± SEM. *P < .05.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal