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. 2020 Jan 30;16(1):e1008261. doi: 10.1371/journal.ppat.1008261

Fig 3. EphA2 is upregulated in human and mouse primary brain endothelial cells in response to inflammatory cytokines.

Fig 3

Representative data showing von Willebrand factor (VWF) immunofluorescence staining (cell nuclei stained with DAPI-blue) (A, left), CD31 flow cytometry staining (gray histogram: isotype control, blue histogram: anti-CD31) (A, right), and transmission electron microscopy (B) of cultured MBMECs isolated from C57BL/6J mice. White arrows in (B) indicate endothelial cell contact points. Scale bars represent 25μm (A) and 0.5μm (B). (C-D) EphA2 transcription in human (C) and mouse (D) BMECs incubated for 24 hours with naïve RBC lysates (nRBC), P. falciparum 3D7-infected RBC lysates (Pf pRBC), PbA-infected RBC lysates (PbA pRBC), or no RBC lysate (∅ RBC) plus human (C) or mouse (D) LT-α, TNF-α, or media (M) (n = 3–6 endothelial preparations/group). Values are normalized to untreated cells. (E) Immunofluorescence images and fluorescence quantification of EphA2 (red) on MBMECs unstimulated or stimulated with PbA-infected RBC lysates (PbA pRBC) in the presence of absence of LT-α for 24 hours. Cell nuclei stained with DAPI (blue). Scale bars represent 25μm. Images representative of two endothelial preparations. (F) LT-α transcription relative to naïve mice (dashed line) in whole brains, livers, lungs, and spleens of C57BL/6J mice (n = 8-9/group) at day 6 post-infection with PbA. (G) EphA2 transcription relative to naïve mice (dashed line) in brains of LT-α-/- (n = 7) and TNFR2-/- (n = 3) mice at day 6 post-infection with PbA compared to wild-type C57BL/6J mice (n = 6–11). (H) LT-α and EphA2 transcription relative to naïve mice (dashed line) in brains of isotype control (n = 9) and anti-TNFR2 (n = 8) treated mice at day 6 post-infection with PbA. Bars in all graphs represent the mean ± SEM. Statistical analyses: Kruskal-Wallis and Dunn’s multiple comparisons tests (C, D, E, F) and Mann-Whitney test (G, H). Only statistically significant (p<0.05) values are shown. Figures represent combined data from 1 (G-right panel), 2 (E, F, G-left panel, H), or 3 (C, D) independent experiments.