Fig. 1.

AJAP1 downregulation increases the sprout formation in the HUVEC sprouting assay. (A) Schematic overview of the siRNA binding sites for AJAP1 mRNA. Three different siRNAs targeting two distinct exons on human AJAP1 mRNA were transfected in HUVECs. Red marks indicate the respective siRNA binding site. Yellow indicates the protein coding region on AJAP1 mRNA. Ex, exon. (B′) The knockdown efficiency of each experiment was determined separately by measuring the relative AJAP1 mRNA expression compared to RPS13 by qRT-PCR. The expression levels were normalized to relative mRNA expression of cells transfected with scrRNA. The plot shows mean±s.d. of the five performed experiments. (B″) AJAP1 downregulation on protein level was confirmed by western blot. The relative amount of AJAP1 to GAPDH is shown. A representative example is shown. (C) Representative samples of sprouting spheroids are shown 24 h after embedment into collagen I. Microscope: Axiovert 40 CFL; objective lens: A-Plan 10×/0.25Ph; scale bar: 50 µm. (D,E). The average cumulative sprout length and the average number of sprouts per spheroid was quantified and is presented in a box and whiskers plot. The box contains 50% of the data points, the middle line of the box is the median and the square is the arithmetic mean. Whiskers represent minimum/maximum values. Per condition, five independent experiments with ten replicates were performed. Statistics was performed using the t-test with post hoc Bonferroni correction for multiple comparison between all conditions.