Fig. 4.

Optogenetic kindlin-2 interaction with integrin β3 induces angiogenic sprout formation by endothelial cells. (A) Representative images and quantification of sprout formation in endothelial cells expressing the indicated optogenetic integrin β3 and kindlin-2 constructs. After overnight incubation of endothelial cells in the dark or in the presence of blue light (0.5 s illumination every 60 s), angiogenic sprouts were quantified. (B) Comparison of sprout formation by endothelial cells expressing either β3ΔRGT–GFP–LOVpep and/or wild-type β3–GFP–LOVpep in fibrin gel bead assay. Note that blue light-induced interaction with ePDZb1–mCherry–kindlin-2 with β3ΔRGT restores endothelial cell sprout formation to a level seen with wild-type type β3, the latter independent of the presence of blue light. Data represent mean±s.e.m. from four experiments. (C) Lentivirus encoding kindlin-2 shRNA (shRNA-1 and shRNA-2) or a control shRNA was used to transduce endothelial cells expressing β3ΔRGT–GFP–LOVpep and ePDZb1–mCherry–kindlin-2. Note on these western blots that shRNA-1 knocked down the expression of endogenous kindlin-2, but not ePDZb1–mCherry–kindlin-2. By contrast, shRNA-2 knocked down both the endogenous and the recombinant kindlin-2. (D) Representative images and quantification of the effects of kindlin-2 shRNA on endothelial cell sprouting. Data represent mean±s.e.m. from three experiments. **P<0.01; *P<0.05 (paired Student's t-test).