Fig. 2.

Roles of nestin-expressing endothelial cells on angiogenesis. A and B Flowcytometric analysis of (A) VEGFR2 or (B) PDGFRβ expression in nestin-expressing and -nonexpressing endothelial cells of a murine lung (n = 5 per group). C (left) Workflow of 3-dimensional (3D) culture to examine the angiogenetic ability in nestin-expressing (Nes⁺) and -nonexpressing (Nes⁻) endothelial cells (EC) obtained from nestin-GFP murine lungs. (middle) Obtained Nes⁺ and Nes⁻ ECs were cultured under 3D culture system supplemented with 50 ng/mL of VEGF or 50 ng/mL of PDGF. Representative results of 3D culture after 21 d. Scale bars = 100 μm. (right) Calculation of the vascular densities using imaging software as described in Method (n = 3 per group). D Nes mRNA expression in each murine cell line: 3T3 (fibroblasts), LA-4 (epithelial cells), and MS1 (endothelial cells) (n = 5 per group). E Effect of gene silencing using nestin siRNA on MS1 (n = 5 per group). F MS1 cells were cultured under 3D culture system supplemented with 50 ng/mL of VEGF or 50 ng/mL of PDGF. Representative results of MS1 treated with control or nestin siRNA after 6 d of 3D culture. Scale bars = 100 μm