N-cadherin–Trio complex increases VE-cadherin recruitment rate at AJs. (A) Confocal images of HPAECs grown on either gelatin or N-cad-BioS after depletion of Trio with siRNA or treatment with control siRNA. Cells were stained for VE-cadherin (green) and nuclei (DAPI, blue). Bar, 10 µm. (B) Quantification of VE-cadherin junction area using images in A. n = 9 images per condition from three independent experiments. ***, P < 0.001, ANOVA with Tukey’s post hoc test. Depletion of Trio significantly reduced VE-cadherin adhesion area only in cells grown on N-cad-BioS. (C) Quantification of VE-cadherin junction area (for cells expressing GFP or GFP-Trio) following overexpression of GFP alone, GFP-Trio-FL, and Trio GEF1 and GEF2 inactive mutants, Trio-D1d and Trio-Dd2, respectively. Loss of VE-cadherin after Trio depletion is rescued by overexpressing FL Trio-GFP and partially with GEF1 but not GEF2 “dead” Trio mutants. n = 12–25 images per condition from three independent experiments. *, P < 0.05; ****, P < 0.0001, ANOVA with Tukey’s post hoc test. (D) Time-lapse images of VE-cadherin–Dendra2 before and after photo-conversion at t = 0 within (as in Fig. 4) in HPAECs grown on N-cad-BioS after depletion of Trio. 488 nm channel (green) = unconverted VE-cadherin; 543 nm channel (red) = photo-converted VE-cadherin. Dashed lines of grayscale images outline the cell borders. Bar, 5 µm; insets, 2 µm; time is shown in minutes. (E and F) Rate of VE-cadherin recruitment to AJs (E) and recruitment rate constant (F) from data in D. n = 7 junctions from three independent experiments. ***, P < 0.001, a two-tailed, unpaired t test. (G and H) Rate of VE-cadherin internalization from AJs (G) and internalization rate constant (H) from data in D. n = 7 junctions from three independent experiments. ns, a two-tailed, unpaired t test. (B, F, and H) Data are shown as mean ± SD. (C, E, and G) Data are shown as mean ± SEM. See also Fig. S3.