Fig. 1

Loss of Rab10 impairs endothelial basement membrane secretion. (a) Immunoblot of Rab10 in ECs transfected with either scrambled or Rab10 siRNA and probed for indicated proteins. (b) Representative images of scramble or Rab10 siRNA-treated ECs and stained for collagen IV (Col IV) (red), actin (green), and DNA (blue). Dotted green line indicates cell outline. Arrowheads denote extracellular Col IV secretion. (c) Graph of Col IV extracellular ratio in scramble or Rab10 siRNA-treated ECs. N number of cells. (d) Representative image of ECs expressing GFP only (control), GFP-Rab10 constitutively active (CA) or GFP-Rab10 dominant negative (DN), stained for Col IV (red) and DNA (blue). Dotted green line indicates cell outline. Arrowheads denote extracellular Col IV secretion. (e) Graph of Col IV extracellular ratio in ECs expressing GFP only (control) or GFP-Rab10 CA/DN. N number of cells. (f) Representative images of scramble or Rab10 siRNA-treated ECs and stained for laminin (red) perlecan (green), actin (grey), and DNA (blue). Dotted green line indicates cell outline. Arrowheads denote extracellular Col IV secretion. (g) Graph of extracellular basement membrane (BM) ratio in scramble or Rab10 siRNA-treated ECs between indicated secreted proteins. N number of cells. (h) Representative image of fibrin-bead sprouts between indicated siRNA treatment groups. Sprouts were stained for actin (grey) to delineate sprout morphology. (i–k) Graphs of sprouting parameters for scramble or Rab10 siRNA-treated sprouts. N number of measurements. (l) Representative images of ECs expressing GFP only (control) or GFP-Rab10 CA/DN in fibrin-bead sprouts. Dotted red line indicates line scan of (m). Arrowheads denote extracellular Col IV secretion. (m) Line scan measurement of Col IV fluorescence intensity shown in (l). (n–p) Graph of sprouting parameters for GFP only (control) or GFP-Rab10 CA/DN expressing sprouts. N number of measurements. For all experiments, data represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p-values. All p-values are from two-tailed Student’s t-test from at least three experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns not significant