Genetic knockdown of eNOS promotes proliferation and migration but inhibits formation of tube-like structures <i>in vitro</i> in HUVECs.

(A) HUVECs were transfected with sieNOS or scrambled control for 24, 48 and 72 hrs and the knockdown effect was confirmed by qPCR (B) western blot. (C) Endothelial cell proliferation was evaluated 24, 48 and 72 hrs post-transfection using proliferation kit and (D) by counting the cells using Cyto Smart cell counter. Each triplicate was counted twice, and average of all triplicates was calculated for each biological replicate. (E) Later, RNA was extracted 24 hrs post-transfection and qPCR for p21 and (F) protein was extracted 24, 48 and 72 hrs post-transfection and immunoblot was performed for ki67, p21 and GAPDH. (G) ENOS-knockdown and control HUVECs were seeded on Matrigel, and pictures were taken 6 hrs post-seeding (tubes are marked by arrows) and (H) then quantification for mesh area and (I) tube-length were performed. (J) Scratch assay was performed in sieNOS and scrambled control-transfected HUVECs and pictures were taken at 0, 8 and 20 hrs and (K) the cell migration was quantified. *p<0.05, **p<0.01, ***p<0.001 vs. Scr Control. N = 3–5 in triplicates.