Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial Nucleoporin93 (Nup93) Maintains Vascular Homeostasis via Sun1-Dependent Regulation of RhoA/ROCK activity

doi: 10.1161/ATVBAHA.126.324964

Figure Lengend Snippet: (A) Nup93 knockdown significantly increases endothelial RhoA protein levels (representative experiment shown in technical triplicates). Quantified in (B, t-test, each dot a biological experiment [averaged technical triplicates] ).(C) Loss of Nup93 also increases GTP-loaded RhoA levels to indicate heightened RhoA activity (t-test, each dot a biological experiment performed in technical duplicates). (D) shNup93-transduced ECs treated with Y-27632 (10µM; 24hrs) exhibit decreased streptavidin staining, less stress fiber formation, and enhanced VE-cadherin junctional intensity. Representative images shown. Scale bars=50µm. Quantified in (E-G, each dot a biological experiment performed in technical triplicates, 2-Way ANOVA followed by Tukey’s ) . ( H) Y-27632 treatment in Nup93-deficient ECs reduces cellular stiffness, as AFM readouts indicate values similar to that of control conditions. (n=3, 8 reads per group/expt, 2-Way ANOVA followed by Tukey’s). (I) Y-27632 treatment in Nup93-depleted ECs partially rescues both phosphorylated and total eNOS levels, whereas Nup93 remains unaffected. Representative experiment shown in technical duplicates. Quantified in (J-L , n=3, 2-Way ANOVA followed by Tukey’s, each dot a biological experiment [averaged technical duplicates] ). (M) Conditioned media from Y-27632-treated Nup93-deficient ECs show a partial albeit significant restoration in secreted NO levels. (n=3, each dot a biological experiment [averaged technical duplicates], 2-Way ANOVA followed by Tukey’s).

Article Snippet: In vitro RhoA activity was determined by measuring the level of guanosine triphosphate (GTP)-bound RhoA in cultured cells according to the manufacturer’s instructions (Cytoskeleton, BK124).

Techniques: Activity Assay, Knockdown, Staining, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial Nucleoporin93 (Nup93) Maintains Vascular Homeostasis via Sun1-Dependent Regulation of RhoA/ROCK activity

doi: 10.1161/ATVBAHA.126.324964

Figure Lengend Snippet: (A) Endothelial knockdown of Nup93 leads to a selective loss in Sun1 protein expression, whereas Sun2 protein levels remain unchanged. Representative experiment shown in technical triplicates. Quantified in (B) and (C). ( t-test, each dot a biological experiment [averaged technical triplicates]) (D) Schematic: Primary human ECs were first transduced with shNup93 (or shEmpty controls) for targeted knockdown of Nup93. Lentiviral-based methods were then used to express a human Sun1-FLAG construct (or Empty) for exogenous Sun1 expression. (E) Immunoblot analyses indicate successful restoration of Sun1 protein levels in Nup93-deficient ECs, as Sun1 levels are comparable to that of healthy control ECs. Sun2 and Nup93 protein levels, however, remain unaffected. RhoA protein levels also remain elevated despite exogenous Sun1 protein. Representative experiment shown with select groups performed in technical duplicates. Quantified in (F-I , n=3, each dot a biological experiment with select groups performed in technical duplicates, 2-Way ANOVA followed by Tukey’s ). (J) RhoA activity is, however, significantly attenuated with Sun1 re-introduction in Nup93 knockdown ECs. (n=3, each dot a biological experiment [averaged technical duplicates], 2-Way ANOVA followed by Tukey’s).

Article Snippet: In vitro RhoA activity was determined by measuring the level of guanosine triphosphate (GTP)-bound RhoA in cultured cells according to the manufacturer’s instructions (Cytoskeleton, BK124).

Techniques: Activity Assay, Knockdown, Expressing, Transduction, Construct, Western Blot, Control