Fig. 7

Fig. 7

Sorbs1 controls EC adhesive properties via RhoGTPases in vitro and in vivo. A Cdc42, Rac1, and RhoA activity in HUVECs transfected with control (Ctl) or Sorbs1 siRNA. Histogram is from Western blot densitometric analysis of three independent pull-down experiments and represents the ratio between bound active- and total amount of each RhoGTPase in the lysate, relative to control cells (***P < 0.001, *P < 0.05, ns = non-significant, Student’s t-test). B Confocal pictures of peripheral F-Actin (phalloidin staining) in Ctl or Sorbs1 siRNA transfected HUVECS treated ( +) or not ( −) with the C3 RhoA inhibitor. Images are shown using an intensity-based look-up table (from blue = low to red = high). Numbers represent the average signal intensity ± SD in each condition. Scale bar represents 50 μM. C Adhesion complexes were analyzed by confocal microscopy after immunostaining of Paxillin and phospho-Paxillin (p-Paxilin) in HUVECs transfected with control or Sorbs1 siRNA. Scale bar represents 20 μm. Nascent adhesions (NA) and focal complexes (Fx) are characterized by their small size, peripheral location and high p-Paxillin/Paxillin ratio content (arrowheads). Larger and more mature focal adhesion (FA) were defined as bigger than 1 μm², and their proportion in each condition was quantified (n = 21; *P < 0.05, Student’s t-test). D Representative micrographs and quantification of adhesion assays performed with HUVECs transfected with control or Sorbs1 siRNA as described in the “Methods” section. Scale bars represent 100 μm (n = 3 independent experiments; **P < 0.01, Student’s t-test). E Representative live confocal images (Z-maximum intensity projection) of emerging filopodia in the CVP of 26 hpf Tg(LIFEACT:mKate2) embryos injected with Ctl or Sorbs1 morpholino at 5 ng/ul. Enrichment of actin at the cell cortex from the boxed area is visualized using a color look-up table (intensity scale). The number of filopodia per 40 μm and the cortical enrichment of the F-actin signal is indicated for both conditions in the table. Scale bar represents 40 μm (n = 7/WT and n = 9/Sorb1 Mo, *P < 0.05, Mann–Whitney U-test). F WT and sorbs1^−/− embryos were treated or not with RhoA inhibitor at 26 hpf, and the percentage of aISV/vISV at 48 hpf was quantified (n = number of embryos; * P < 0.05; ns = non-significant; χ² with Yates correction). G Quantification of PL number in 10 somites at 54 hpf in WT and sorbs1^−/.⁻ embryos injected with RhoA inhibitor at 26 hpf or left untreated (n = number of embryos; *P < 0.05; ns = non-significant; Mann–Whitney U-test)