Fig. 4

circFIRRE can induce angiogenesis in vitro, ex vivo and in vivo. A Schematic description of the flow of magnetic bead cell sorting. B Validation of differentially expressed circFIRRE in primary endothelia cells by RT-qPCR (n=3 in each group). C Validation of differentially expressed circFIRRE in different endothelia cell lines by RT-qPCR (n=3 in each group). D HUVEC cells were transiently transfected with circFIRRE-siRNAs or scramble siRNA (si-N.C.). 48 hours after transfection, the relative circFIRRE expression in HUVEC cells were detected by RT-qPCR (n=3 in each group). E-F Tube formation assay was applied to determine cell tube formation ability after circFIRRE silencing in HUVEC cells. Scar bar=1mm. Image quantification were conducted as described in methods (n=3 in each group). G Representative immunofluorescence staining of aortic rings. BS1 lectin-FITC (green) indicated endothelial sprouts; α-SMA-Cy3 (red) stained supporting cells; DAPI-stained nuclei (blue). Scale bars=400 μm and 150 μm. H Representative images of CAM photographed on plastic dished after resection from eggs. Scale bars=4 cm and 2 cm. I-L Tube formation, aortic ring and CAM assays were conducted to examine angiogenesis level induced by circFIRRE overexpression, respectively. Values are presented as mean ± SD; the bar charts, error bars and dots represent the quantitative analysis of 3 independent experiments; (B, 2-tailed Student t test; C, D, one-way ANOVA; F, J, two-way ANOVA); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001