Snail knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.

Abstract

<p>(A) Whole flat-mount images showing the colocalization of Snail and VEGFR3. The immunoreactivity of Snail (green) was observed in the sprouting vessel from the vein in P8 retinal vessels. VEGFR3 immunoreactivity (magenta) was also found in the vein. V, vein. (B) Confocal images of iB4 combined with VEGFR3 staining in shCon or shSnail lentivirus-infected retinas at P8. Mice were consecutively injected intraperitoneally with the shCon or shSnail lentivirus at P6 and P7, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g006" target="_blank">Fig 6A</a>. The shSnail lentivirus was the same virus that was described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g001" target="_blank">Fig 1F</a> (shSnail#2). Images of vertical vessels from superficial plexus were taken. Arrows indicate sprouting vessels from veins. Broken line indicates the position of veins in the superficial plexus. Bar, 100 μm. (C) Confocal images of iB4 combined with VEGFR3 staining in the region of the deep capillary plexus in siCon- or siSnail-injected mice at P11. SiRNA injections were performed, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g006" target="_blank">Fig 6A</a>. Cell nuclei were stained with DAPI (blue). The immunoreactivity of iB4 and VEGFR3 was weaker in siSnail-injected mice than in siCon mice. Bar, 100 μm. (D) Quantification of total vessel area and VEGFR3-positive regions in the deep plexus at P11. Over six fields were analyzed. *, p<0.01. (E) Fibrin bead assay. HRECs were transfected with mock and Snail in a combination with VEGFR3 siRNA (siVEGFR3). Representative spheroids are shown for each condition (left). Sprouting numbers per bead, sprouting lengths from each bead, and branch numbers were calculated to quantify endothelial sprouting (right). *, p<0.01.</p