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Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components

Fig 6

DENV2 NS1 induces degradation of sialic acid, activation of cathepsin L, and shedding of heparan sulfate in HMEC-1 in vitro.

(A-D) HMEC-1 monolayers treated with 5 μg/ml of DENV2 NS1 (middle column) or 5 μg/ml of DENV2 NS1 and an inhibitor cocktail (Zanamivir, 100 μM; Cathepsin L Inhibitor, 10 μM; OGT 2115, 1.0 μM; right column). Untreated monolayers were used as a control (left column). Six hours post-treatment, cells were stained for (B) sialic acid (WGA-A647, red; top row images), (C) cathepsin L activity (Magic Red Cathepsin L detection kit, red; middle row images), or (D) heparan sulfate (Heparan Sulfate mAb clone F58-10E4, green; bottom row images) and imaged on a Zeiss LSM 710 Axio Observer inverted fluorescence microscope equipped with a 34-channel spectral detector at 20x magnification. (A) Images were acquired using the Zen 2010 software (Zeiss). Nuclei were stained with Hoechst (blue). Images shown at 20X; scale bar, 10 μM. Representative images shown. (B-D) Quantification of MFI in Fig 6A.

Fig 6