Figure 5

Effect of miRNAs on tube formation in HDLEC. (A, D) HDLEC were transfected with 20 nM each of the miRNA mimics, miRNA inhibitor and/or siRNA. After 48 h, the HDLEC cells (7 × 10³ cells/well) were plated on ECMatrix-coated tissue culture plates in endothelial culture medium including 10 ng/mL basic fibroblast growth factor, 5 ng/mL epidermal growth factor, 20 ng/mL insulin like growth factor 1, and 0.5 ng/mL VEGF. Capillary-like structures within the Matrigel layer were photographed after 4–6 h under a microscope (×50). Defective tube formation points are marked with black arrows. (B) Results similar to those in panel A were obtained in two additional independent experiments, and tube formation was quantified using the ImageJ software. Means were calculated using data from all three independent experiments and expressed as ratios to the value obtained from the scrambled control-transfected HDLEC. (C) Effects of Prox1 siRNA on the expression of Prox1 protein. HDLEC were transfected with 20 nM Prox1 siRNA or the scrambled control. Cells were cultured for 48 h and then harvested for analysis. Western blotting was conducted using three sets of independently transfected HDLEC. Bands were quantified using Fujifilm Multi Gauge software (version 3.0). Means were calculated using data from all three independent experiments and are expressed as ratios to the value obtained from the scrambled control-transfected HDLEC. (E) Results similar to those in panel D were obtained in two additional independent experiments, and tube formation was quantified using ImageJ software. Means were calculated using data from all three independent experiments and are expressed as ratios to the value obtained from the scrambled control-transfected HDLEC. Error bars indicate SDs. *P < 0.05. †P < 0.01.