Fig. 6. NFATc2 regulates VEGF-A isoform-specific endothelial cell migration but not tubulogenesis.

(A–F) Control, scrambled or NFATc2-specific siRNA duplex-treated endothelial cells were seeded into assays to assess endothelial cell (A–C) migration or (D–F) tubulogenesis. (A) Endothelial cells seeded into Transwell filters were stimulated with 0.25 nM VEGF-A₁₆₅ or VEGF-A₁₂₁ for 24 h before being fixed and stained with 20% (v/v) crystal violet. Scale bar, 1000 µm. (B,C) Quantification of endothelial cell migration compared to (B) non-transfected or (C) individual controls. Error bars indicate ±SEM (n≥3). (D) Endothelial cells subjected to different siRNA treatments were co-cultured on a bed of primary human fibroblast for 7 days and stimulated with 0.25 nM VEGF-A₁₆₅ or VEGF-A₁₂₁. Co-cultures were fixed and endothelial tubules were stained and visualised using an anti-PECAM1 antibody followed by fluorescent secondary antibody. Scale bar, 1000 µm. (E,F) Quantification of endothelial cell tubulogenesis including total (E) tubule length or (F) number of branch points. Error bars indicate ±SEM (n≥3). p<0.05 (*), p<0.01 (**), NS = non-specific.