ZO-1 down-regulation reduces endothelial cell migration and angiogenic potential. (A) HDMEC were transfected with the indicated siRNAs for 48 h. Scratch wounds were then inflicted to induce cell migration. Representative images of wounds are shown that were taken at 0 and 16 h after scratching. (B) Percentages of the areas of closure of three independent experiments; shown are means ± 1 SD (error bars). (C–G) The effect of the depletion of ZO-1 on endothelial angiogenic potential was tested in vitro using an MC-based fibrin gel angiogenesis assay (C–E) or Matrigel plugs in vivo (F and G). For in vitro assays, HDMEC were seeded onto beads 24 h after siRNA transfection and were then embedded in a 3D fibrin gel after another 24 h. (C) Sprouting was then analyzed after 4 d by phase-contrast microscopy. (D) The cells were then fixed, permeabilized, and stained for nuclei (blue) and F-actin (red) to monitor coverage of beads with cells. (D and E) The number of sprouts per bead (D) and the mean length (E) were then determined (shown are means ± 1 SD [error bars] of four experiments). (F and G) For in vivo assays, mice were injected with Matrigel-containing FGF and siRNAs as indicted to induce angiogenesis. After 7 d, the plugs were harvested and fixed, embedded in paraffin, and sectioned. Sections in E were stained by hematoxylin and eosin, and were used to quantify the number of vessels shown in F (shown are means ± 1 SD [error bars]; control and FGF, n = 6; siRNA samples, n = 12). Bars, 250 µm.