Supplemental Material:Supplemental Figures S1-S5, Supplemental Videos S1-S5

Supplemental Figure S1. Representative images for (A) primary antibody (1o ab) validation of heparan sulfate (HS) and (B) for wheat germ agglutinin (WGA) staining in 3D endothelium. Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs). Magnification: ×10; scale bar: 500 μm, (n=2-3).Supplemental Figure S2. Cytotoxicity effect of TNF-α on 3D endothelium. Lactate dehydrogenase (LDH) activity in 3D HUVECs following treatment of either vehicle (Veh) or 10 ng/ml TNF-α for 24 h. Statistical analysis was determined by Student’s t-test (*pSupplemental Figure S3. Representative fluorescence images and quantification of mean fluorescence intensity (MFI) per cell of (A) heparan sulfate (HS) and (B) EGX components stained with wheat germ agglutinin (WGA) after 3D HUVECs were stimulated with Veh or 1 ng/mL TNF-α for 5 h. Magnification: ×10; scale bar: 500 μm. Student’s t-test was used to compare Veh and TNF-α (*p Supplemental Figure S4. Cytotoxicity effects of NS1 on 3D endothelium. Lactate dehydrogenase (LDH) activity in 3D HUVECs treated with vehicle (Veh) or NS1 for 12 h. Statistical analysis was determined via Student’s t-test (ns = not significant). Data are presented as means ± SDs (n = 7).Supplemental Figure S5. Representative bright-field images of 3D HCAECs from Day 0 to Day 5. HCAECs were cultured in two-lane Organoplates (Mimetas), forming 3D tubular structures. Magnification: ×5; scale bar: 200 μm.Supplemental Video S1. Characterization of 3D endothelium. Human umbilical vein endothelial cells (HUVECs) were cultured in two-lane Organoplates (Mimetas), forming 3D tubular structures within 4–5 days. The 3D HUVECs were stained for CD31 (green), F-actin (red), and nuclei (blue). The 3D architecture was visualized using Z-plane images captured at 8-μM intervals in confocal mode with the Opera Phenix High-Content Screening System.Supplemental Video S2 and S3. Comparisons of heparan sulfate (HS) expression between 2D endothelial cell cultures and 3D endothelium. Human umbilical vein endothelial cells (HUVECs) were cultured in (S2) 24-well plates until 95% confluency or (S3) two-lane Organoplates to generate 3D endothelium. They were stained for HS (green) and nuclei (blue). Images were captured using Z-plane images at 8-μM intervals in confocal mode via the Opera Phenix High-Content Screening System.Supplemental Video S4 and S5. Comparisons of wheat germ agglutinin (WGA) staining between 2D endothelial cell cultures and 3D endothelium. Human umbilical vein endothelial cells (HUVECs) were cultured in (S4) 24-well plates until 95% confluency or (S5) two-lane Organoplates to generate 3D endothelium. They were stained with WGA (green) and nuclei (blue). Images were captured using Z-plane images at 8-μM intervals in confocal mode via the Opera Phenix High-Content Screening System.