Fig. 3.

Loss of EROS disrupts endothelial cell function

A-C. Representative images of siRNA transfected HUVEC with either siControl, siRAC1 or siEROS show staining of F-actin binding fluorescent dye conjugated phalloidin (green) and nuclear counterstain with DAPI (blue). Scale bar is 10 μm. D. Quantitative analysis of intact actin filaments as performed shown in Figs. S5A and B indicates disorganized cytoskeleton in HUVEC downregulating EROS (yellow plot, n = 20) as well as RAC1 (blue plot, n = 26) versus siControl (green plot, n = 25). E-F. Statistical evaluation of scratch area recovery in Control (E) or under VEGF-treated conditions (F) indicates lower migratory activities in HUVEC following knockdown of RAC1 (blue plots) as well as EROS (yellow plots) versus siControl (green plots). G. CAS9 (Control) and EROS SG1 or SG2 transduced HUVEC were seeded to ∼20 % confluence in endothelial cell growth medium and monitored at day 0, 2, 4 and 6. Graph shows progression of cell proliferation in Control cells (black line, n = 8), while both EROS SG populations (dark and light gray lines, n = 8 each) over time even decreased in cell number counted per mm². H. Statistical analysis of fluorescence-based β-galactosidase activity indicates similar degree of senescence progression in EROS SG1 (blue plot, n = 11) as well as EROS SG2 (yellow plot, n = 11) compared to CAS9 infected cells (Control, green plot, n = 11). For all panels when indicated, ***P < 0.001 and ****P < 0.0001 (1way ANOVA for equal variance). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)