Fig 5. Addition of clathrin-mediated endocytosis inhibitors prevents EGL disruption and endothelial hyperpermeability triggered by DENV NS1.

(A) The integrity of the EGL on HPMEC was assessed by the presence of heparan sulfate [9] surface expression (green, bottom row), as well as cathepsin L (CTSL) activity (red, top row), at 6 hpt with DENV NS1 (5 μg/ml) in the presence or absence of Pitstop 2 (12.5 μM), or Pitstop 2 alone, at 37°C, as visualized via IFA. Nuclei were stained with Hoechst (blue). Images (20X; scale bars, 50 μm) are representative of 3 independent experiments. (B) Quantification of MFI in (A) from 3 independent experiments expressed as either fold change of cathepsin L activity from Pitstop 2 control values (left) or normalized percentage of heparan sulfate from Pitstop 2 control values [10]. Error bars indicate SEM. **, p<0.01; ***, p<0.001. (C) HPMEC were grown on Transwell semi-permeable membranes (0.4 μm pore size), and the apical chamber was treated with either DENV NS1+ (5 μg/ml) and DMSO (blue squares); the dynamin inhibitor Dynasore (25 μM) alone (purple diamonds) or Dynasore and NS1 (orange triangles); or the clathrin-mediated endocytosis inhibitor Pitstop 2 (12.5 μM) alone (light green triangles) or Pitstop 2 and NS1 (pink circles). A TEER assay was used to evaluate the effect of NS1 and inhibitors on endothelial permeability at indicated time-points over 48 hours. (^) represents change of medium. Relative TEER values from 2 independent experiments performed in duplicate are plotted. Error bars indicate standard error of the mean (SEM).