Fig 2: EphB4-induced signaling in cultured ECs.(A) Western blot analysis of lysates from cultured HUVECs stimulated with control human IgG/Fc (Ctrl Fc), human ephrin-B2/Fc (B2/Fc) or mouse EphB4/Fc (B4/Fc) (4 µg/ml, preclustered with 10µg/ml goat anti-human IgG), concurrently treated with inhibitors (LY294002, U0126, PP2) or DMSO for 30 min. Active Src (pSrc) was determined by anti-Src immunoprecipitation (IP) followed by anti-Src or anti-phosphotyrosine (4G10) immunoblotting. Bottom panel indicates Src input in total cell lysate (TL). Molecular weights (kDa) are indicated. N = 3 for all treatments. (B) Graphs show relative quantitation of pAkt/Akt, pERK1/2/ERK1/2, pSrc/Src and pSrc/Src/input. N = 3 for LY294002 and U0126 experiments. For PP2 experiment N = 4 for Ctrl Fc/DMSO, B4/Fc/DMSO and B4/Fc/PP2 and N = 5 for all the other conditions. Data represented as mean ± s.e.m. P values calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. (C) Colocalization (arrowheads) of EphB4 (green) and phospho-CAV1 (pCAV1, red) in HUVECs 30 min after stimulation with human IgG/Fc or ephrin-B2-Fc. Rightmost panels show higher magnification of selected areas. Cell junctions, VE-Cadherin (CDH5, white); nuclei, DAPI (blue). ns, not significant.Figure 6—source data 1.Source data for Figure 6B.

Fig 3: FA-mediated traction forces drive flow-induced polarity(A) Representative image of AJs (VE-cadherin [VEcad], blue) and FAs (vinculin [green] and ITGA5 (red) in static or flow-stimulated HUVEC monolayers (high magnification from Figure S5C).(B) Box plot for the number of FAs in static or flow conditions. n = 8 (static) and n = 6 (flow). p value from Mann-Whitney test. Whiskers: min. to max.(C) Representative super-resolution image of co-localization between vinculin (green) and activated integrin beta 1 (aITGB1) (red) in static or flow-stimulated HUVEC monolayers. Scale bars: 2 µm.(D) Top: angular histograms for the distribution of vinculin FA orientation in static and flow conditions. Bottom: mean angle of orientation of the vector from vinculin to aITGB1 centroids in relation to right-to-left slide axis in static and flow conditions. Flow direction is right to left. n = 20 images from 4 separate experiments.(E) Box plot for polarity index in static or flow-stimulated HUVEC monolayers treated with PBS (n = 3), RGDS (n = 5), and cilengitide (n = 3), p values from one-way ANOVA with Sidak test. Whiskers: min. to max.(F and G) Mean traction maps (F) and box plot of mean traction forces (G) exerted by static or flow-stimulated HUVEC monolayers. n = 4 static; n = 5 flow. p values from Mann-Whitney test. Whiskers: min. to max.(H) Box plot for numbers of focal adhesions in flow-stimulated HUVEC monolayers treated with DMSO (n = 9), Y-27632 (n = 5), or BBS (blebbistatin, n = 5). p values from Mann-Whitney test. Whiskers: min. to max.(I) Box plot of polarity index in static or flow-stimulated HUVEC monolayers treated with DMSO (n = 4), Y-27632 (n = 5) or BBS (blebbistatin, n = 6). p values from one-way ANOVA with Sidak test. Whiskers: min. to max.

Fig 4: No influenza A virus infected endothelial cells in epithelial–endothelial co-cultures when inoculated directly.(A, B) Well-differentiated airway organoids in co-cultures with lung microvascular endothelial cells were inoculated with mock or (B) pH1N1, H1N1, and H3N2 at MOI 1. At 24 h post-inoculation, the endothelial cells were directly inoculated with homologous viruses. The cells were fixed after 24 h post second inoculation and stained for the endothelial cell marker vascular endothelial-cadherin (VE-CAD magenta) and influenza A virus nucleoprotein (NP, green). Hoechst (blue) was used to visualize nuclei. Scale bar: 20 µM.

Fig 5: Abortive infection of influenza A virus in lung microvascular endothelial cells (LMECs).(A, B) To evaluate replication efficiency, LMECs were plated on the (A) apical or (B) basolateral side of a transwell filter. LMECs were inoculated with pH1N1, H1N1, or H3N2 virus at MOI 1, and at the indicated timepoints supernatants of the apical and basolateral sides were harvested, and virus titers were determined by endpoint titration. Infection efficiency was determined by immunofluorescence staining. (C, D) LMECs plated on the (C) apical and (D) basolateral sides were inoculated with pH1N1, H1N1, or H3N2 virus. Cells were fixed 24 h post-inoculation and stained for the endothelial cell marker vascular endothelial-cadherin (VE-CAD, magenta) and influenza A virus nucleoprotein (NP, green). Hoechst (blue) was used to visualize nuclei. (E) Percentage of infection determined by flow cytometry at 24 and 72 h post-inoculation. (F) Intracellular viral RNA genome copies were quantified by quantitative real-time PCR at indicated timepoints. Data represent mean ± SD from at least three independent experiments performed in biological duplicates, and flow cytometry was performed in biological triplicates. A one-way ANOVA multiple comparison test was used to compare groups (*< 0.05, **<0.01, ***<0.005). Scale bar: 20 µm.