Untreated cells were used like a control for basal Neu2 expression

Untreated cells were used like a control for basal Neu2 expression. surface and DENV2 NS1 binding to HPMEC monolayers in the presence of different concentrations of DENV2 NS1 (1.25, 2.5, 5, and 10 g/ml) in Fig 2C. Results represent imply fluorescence intensity (MFI) ideals from three self-employed experiments. Grey bars represent relative sialic acid manifestation, determined by normalizing each condition to untreated control cells (relative sialic acid manifestation = sialic acid manifestation in DENV2 NS1-treated monolayers/sialic acid manifestation in untreated control monolayers). The blue collection represents DENV2 NS1 binding.(TIF) ppat.1005738.s001.tif (1.5M) GUID:?16B181A1-9EEE-4EC7-8544-A34D8AD430F6 S2 Fig: Related to Fig 1: DENV2 NS1-induced endothelial hyperpermeability is not a result of LPS contamination and occurs in additional endothelial cell types. (A) TEER assay to evaluate the effect of DENV2 NS1 (5 g/ml) on HMEC-1 endothelial permeability. Relative TEER values from one self-employed experiment performed in duplicate are plotted in the indicated time points. Error bars indicate standard error of the mean (SEM). DENV2 induces statistically significant decreases in TEER (p 0.05). (B) Effect of polymyxin B on DENV2 NS1-mediated endothelial hyperpermeability (TEER) in HPMEC monolayers. Relative TEER ideals from three self-employed experiments performed in duplicate are plotted at indicated time points (hpt). TEER ideals for monolayers treated with DENV2 NS1 combined with polymyxin B (25 g/ml) are not significantly different from monolayers treated with DENV2 NS1 only. Error bars show SEM throughout.(TIF) ppat.1005738.s002.tif (532K) GUID:?C7A7D414-E620-4646-A7A5-A46AA4B1570F S3 Fig: Related to Fig 2: The lectin WGA primarily binds to sialic acid on the surface of HPMEC, and DENV2 NS1 disrupts Sia expression about the surface of different endothelial cells. (A) Sia manifestation on HPMEC monolayers after treatment with recombinant neuraminidase from (0.5 UI), examined by confocal microscopy. Sia was stained with WGA-A647 (reddish) at indicated time points (hpt). Untreated cells were used like a control for basal Sia manifestation. Sarolaner Nuclei stained with (blue). Images (20X) are representative of three self-employed MDK experiments. Scale pub, 10 M. (B) Quantification of MFI in S3A Fig from three self-employed experiments. Sia manifestation in monolayers treated with recombinant neuraminidase is definitely significantly different than in untreated control monolayers (p 0.0001). (C) Sia manifestation on HUVEC Sarolaner monolayers after treatment with DENV2 NS1 (5 g/ml), examined by confocal microscopy. Sia was stained with WGA-A647 (reddish) at indicated time points (hpt). Nuclei are stained with (blue). Untreated cells were used as control for Sarolaner basal Sia manifestation. Images are representative of one individual experiment (20X). (D) Sia manifestation on HMEC-1 monolayers after treatment with DENV2 NS1 (5 g/ml), examined by confocal microscopy. Sia was stained with WGA-A647 (reddish) at indicated time points (hpt). Nuclei are stained with (blue). Untreated cells were used as control for basal Sia manifestation. Images are representative of one individual experiment (20X).(TIF) ppat.1005738.s003.tif (2.3M) GUID:?191C2447-0B73-42A5-9FC3-C282DE3F001E S4 Fig: Related to Fig 3: DENV2 NS1 increases the surface staining of syndecan-1 and perlecan in the EGL of HPMEC. (A) Quantification of syndecan-1 MFI in Fig 3A. Staining is definitely significantly higher with DENV2 NS1 compared to settings at 0.5C12 hpt (p 0.0001). Error bars show SEM. (B) Manifestation of perlecan (green) on the surface of HPMEC monolayers over time (hpt) after treatment with DENV2 or WNV NS1 proteins (5 g/ml), examined by confocal microscopy. Untreated cells were used like a control for basal perlecan manifestation. Nuclei are stained with (blue). Images are representative of three individual experiments (20X). Level pub, 10 M. A trace of the MFI of one representative field is definitely demonstrated below each image.(TIF) ppat.1005738.s004.tif (1.9M) GUID:?82DCB8FF-99AD-458B-BB8F-5C1511F6BA98 S5 Fig: Related to Fig 5: DENV2 NS1 increases cathepsin L activity in both HMEC-1 and HUVEC monolayers. (A) Cathepsin L proteolytic activity (Magic Red assay, in reddish) in HUVEC monolayers over time (hpt) after treatment with DENV2 NS1 (5 g/ml). Nuclei are stained with (blue). Untreated cells were used as Sarolaner control for basal cathepsin L manifestation. Images are representative of one individual experiment (20X). (B) Cathepsin L proteolytic activity (Magic Red assay, in reddish) in HMEC-1 monolayers over time (hpt) after treatment with DENV2 NS1 (5 g/ml)..