Figure 6.

RUNX3 transcript variant’s effect on the angiogenic effect of EOC cell supernatant. (A) Using conditioned media (CM) derived from A2780 cells, considerable amounts of HUVEC sprouting was detected. CM of native A2780 alone led to a 14-fold increase in the amount (Supplementary Figure S5) and a 2-fold increase in sprout length resulting in a 30-fold higher effect compared to RPMI + 2% FSC control. The overexpression RUNX3 had no effect on the sprouting compared to the native A2780 cells. After the addition of 10 ng/mL vascular endothelial growth factor (VEGF), the CM effect (3-fold increase) was still visible. n_spheroid ≥ 7. (B) The CM from A13-2-12 cells showed a reduced angiogenic potential similar to the control. The addition of 10 ng/mL VEGF led to a significant increase in formed sprouts. While induction of human umbilical vein endothelial cells (HUVEC) sprouting was seen upon incubation with CM from RUNX3 TV1 expressing A13-2-12 cells, the effect was not significant compared to the empty vector control. The reduction of the sprouting caused by RUNX3 TV2 CM was also not statistically significant, while the difference between the two variant CM did differ. n_spheroid ≥ 8. * p < 0.05, *** p < 0.001, **** p < 0.0001 in 2-way ANOVA. (C,D) Normalized raw intensity data from the Proteome Profiler™ array. Only proteins with signals above the background are shown. Based on current publications, the regulators were either defined as an activator (red), inhibitor (blue) or indifferent (gray). Signals were normalized against the raw intensity of the reference spots on each blot, respectively. Signal quantification of A2780 CM showed more activators than inhibitors, and specifically, high amounts of VEGF were detected (C). Quantification of signals from A13-2-12 CM showed higher signal intensities of inhibitory proteins (D).