Panel a shows a Western blot analysis, with bands for VE-cadherin, SNX9, and beta-actin. Panel b presents a bar graph quantifying VE-cadherin and SNX9 protein levels normalized to beta-actin, with the vertical axis labeled Relative protein expression (arbitrary units) and the horizontal axis labeled shControl, shSNX9-2, and shSNX9-3. Panel c displays confocal images of shControl and shSNX9 HUVECs immunostained for VE-cadherin, highlighting asymmetric junctions with ROI marked. Panel d features a bar graph showing the percentage of straight or bifurcated asymmetric adherens junctions for shControl and shSNX9 HUVECs, with the vertical axis labeled Percentage of asymmetric adherens junctions and the horizontal axis labeled shControl and shSNX9. Panel e includes still images from time-lapse imaging of shControl and shSNX9 BOECs overexpressing VE-cadherin-GFP. Panel f shows a dot plot indicating maximum asymmetric adherens junction length in micrometers during time-lapse imaging of shControl and shSNX9 BOECs, with the vertical axis labeled Asymmetric adherens junction length (micrometers) and the horizontal axis labeled shControl and shSNX9. Panel g presents a dot plot indicating asymmetric adherens junction lifetime in minutes during time-lapse imaging of shControl and shSNX9 BOECs, with the vertical axis labeled Asymmetric adherens junction lifetime (minutes) and the horizontal axis labeled shControl and shSNX9. Panel h shows a Western blot analysis of control and DNM2-depleted HUVECs, with bands for dynamin-2 and beta-actin. Panel i features a bar graph quantifying dynamin-2 protein levels normalized to beta-actin, with the vertical axis labeled Relative protein expression (arbitrary units) and the horizontal axis labeled shControl, shDNM2-1, and shDNM2-2. Panel j includes still images from time-lapse imaging of shControl and shDNM2-1 HUVECs overexpressing VE-cadherin-GFP. Panel k shows a dot plot indicating maximum asymmetric adherens junction length in micrometers during time-lapse imaging of shControl and shDNM2-1 HUVECs, with the vertical axis labeled Asymmetric adherens junction length (micrometers) and the horizontal axis labeled shControl and shDNM2-1. Panel l presents a dot plot indicating asymmetric adherens junction lifetime in minutes during time-lapse imaging of shControl and shDNM2-1 HUVECs, with the vertical axis labeled Asymmetric adherens junction lifetime (minutes) and the horizontal axis labeled shControl and shDNM2-1. Panel m features a bar graph quantifying Golgi orientation in the first four rows of follower cells at the scratch edge of shControl and shDNM2-1 HUVECs, with the vertical axis labeled Percentage of Golgi oriented toward the leading edge and the horizontal axis labeled shControl and shDNM2-1. Panel n shows a bar graph quantifying endothelial wound closure 9 hours after scratch of shControl, shDNM2-1, shSNX9-3, and simultaneous shDNM2 plus shSNX9 HUVECs, with the vertical axis labeled Wound closure (percentage) and the horizontal axis labeled shControl, shDNM2-1, shSNX9-3, and shDNM2 plus shSNX9.
SNX9 controls VE-cadherin turnover at asymmetric junctions. (a) Representative Western blot analysis of control and SNX9-depleted HUVECs using indicated shRNAs blotted for VE-cadherin, SNX9, and β-actin. (b) Quantification of VE-cadherin and SNX9 protein levels normalized to β-actin, from three independent experiments. (c) Confocal images of shControl and shSNX9 HUVECs immunostained for VE-cadherin highlighting AAJs. Black arrows in the ROI highlight representative bifurcated AAJs, where VE-cadherin staining diverges from the main junctional direction. (d) Bar graphs displaying the percentage of straight or bifurcated AAJs for shControl and shSNX9 HUVECs. n = 417 and 520 AAJs in shControl and shSNX9 ECs, respectively, from three independent experiments. (e) Stills from a widefield time-lapse imaging sequence of shControl and shSNX9 BOECs overexpressing VE-cadherin–GFP (See Video 11). (f) Dot plots indicating maximum AAJ length in µm during time-lapse imaging of shControl and shSNX9 BOECs. n = 75 and 84 AAJs for shControl and shSNX9 ECs, respectively, from three independent experiments. (g) Dot plots indicating AAJ lifetime from start to end of AAJ formation and disappearance in minutes during time-lapse imaging of shControl and shSNX9 BOECs. n = 75, 84 AAJs for shControl and shSNX9 ECs, respectively, from three independent experiments. (h) Representative western blot analysis of control and DNM2-depleted HUVECs using indicated shRNAs blotted for dynamin-2 and β-actin. (i) Quantification of dynamin-2 protein levels normalized to β-actin, from three independent experiments. (j) Stills from a widefield time-lapse imaging sequence of shControl and shDNM2-1 HUVECs overexpressing VE-cadherin–GFP (See Video 12). (k) Dot plots indicating maximum AAJ length in µm during time-lapse imaging of shControl and shDNM2-1 HUVECs. n = 76, 75 AAJs for shControl and shDNM2-1 ECs respectively, from three independent experiments. (l) Dot plots indicating AAJ lifetime from start to end of AAJ formation and disappearance in minutes during time-lapse imaging of shControl and shDNM2-1 HUVECs. n = 76, 75 AAJs for shControl and shDNM2 ECs, respectively, from three independent experiments. (m) Quantification of Golgi orientation in the first four rows of follower cells at the scratch edge of shControl and shDNM2-1 HUVECs. For each shRNA, at least n = 7 images per experiment were quantified from three independent experiments. (n) Quantification of endothelial wound closure 9 h after scratch of shControl, shDNM2-1, shSNX9-3, and simultaneous shDNM2 + shSNX9 HUVECs. At least n = 2 movies per experiment were quantified from three independent experiments. Data are presented as mean ± SD of experiment means (b, f, g, i, k, l, m, and n) or SEM (d). Circles indicate individual data points, and triangles indicate means of individual experiments with colors corresponding to the experimental replicates. Statistical analysis was performed using one-way ANOVA in b and i, followed by Tukey’s multiple comparisons in n, a χ-square test in d, an unpaired two-sided Student’s t-test (m), and Mann–Whitney U test in f, g, k, and l. P values are indicated on the graphs. Scale bars = 30, 10, and 5 μm. Source data are available for this figure: SourceData F6.