Figure 4

SPRY1 silencing protects cells from apoptosis and induces endothelial cell adhesion, migration, and tube formation. A. SPRY1 mRNA levels quantified by qRT-PCR from ABAE cells transfected (48 h) with non-silencing siRNA (Control) or with two different SPRY1 siRNAs. B. SPRY1 protein level measured by Western blotting from ABAE cells transfected as indicated in A. The beta-tubulin level was also measured as an internal control. Bottom panel: quantification of the top panel: SPRY1/Beta tubulin. C. Apoptosis was assessed 48 hours after ABAE cell transfection with non-silencing (Control) or with a SPRY1 siRNA by quantification of caspase-3 activity. D. Effect of SPRY1 silencing on adhesion of ABAE cells to fibronectin or vitronectin. E. Migration in a modified Boyen chamber assay. 36 h after transfection cells were seeded in the upper chamber of the Boyden. Migration was assessed by counting cells on the lower face of the membrane after 16 h. Arrow head: pore of the membrane and arrow: cells stained with crystal violet. F. 36 h after transfection, ABAE cells were seeded on Matrigel and incubated for 16 h. Living cells were labeled with calcein-AM. Quantitative analysis of network structures was performed by measuring the number of intersections between tubes in the network (arrows). E, F: Top panel: representative images for migration and tube network formation assays. Bottom panel: Quantification. A-F: Data are mean fold + SD (n = 3). The results are representative of at least three independent cell transfections. *: significant at p < 0.05.