Extended Data Fig. 5: Antibody neutralisation or siRNA depletion of SDC4 in hCMEC/D3 cells induces the loss of cell integrity in response to meningococcal infection.
From: Angiopoietin-like 4 protects against endothelial dysfunction during bacterial sepsis
a-e, hCMECs/D3 cells were either uninfected or infected with N. meningitidis 2C4.3 for 30 min and treated with 10 μg.mL−1 of anti-ANGPTL4 or anti-SDC4 antibodies for 2 h and 30 min. a, Cells were fixed and stained for VE-cadherin, Actin and DAPI and analysed by fluorescent microscopy. Representative images of 3 independent experiments are shown (scale bars, 50 μm). b, c, Quantifications of VE-cadherin enrichment at intercellular junctions and of intercellular spaces area were performed with ImageJ software. Error bars show mean ± s.e.m. (n = 15 fields from n = 2 independent experiments); Ordinary one-way ANOVA Tukey’s multiple comparison test ****P < 0.001; NS, not significant (NS1 = 0.9978, NS2 = 0.6648). d, Apoptosis assay. Error bars show mean ± s.e.m. (n = 2 independent experiments performed in triplicate). Ordinary one-way ANOVA Tukey’s multiple comparison test performed at 9 h compared to the uninfected conditions: *P < 0.05, NS, not significant (P > 0.05; NS1 = 0.9372, NS2 = 0.9941); or compared to the infected condition: § P < 0.05, NS, not significant (P > 0.05; NS3 = 0.9911). e-i, hCMEC/D3 cells were transfected with small interfering RNA (siRNA) against ANGPTL4, SDC4, or SDC1 or with a non-targeting control siRNA. 48 h post-transfection, cells were either uninfected or infected with N. meningitidis 2C4.3 for 30 min and treated with 1 μg.mL−1 of rhANGPTL4 or vehicle for 2 h and 30 min. e, ANGPLT4 depletion efficiency was evaluated by measuring the release of soluble ANGPTL4 in the cell supernatants. Error bars show mean ± s.e.m. from one experiment performed in duplicate. f, SDC4 and SDC1 depletion efficiency was evaluated by immunoblotting using clathrin as a loading control. Bottom: shown are the ratio between SCD4 and clathrin signals. g, Cells were fixed and stained for VE-cadherin, bacteria and DAPI and analysed by fluorescent microscopy. The images are representative of VE-cadherin staining in two independent experiments performed in duplicate (scale bars, 50 μm). h, Quantifications of VE-cadherin enrichment at intercellular junctions were performed with ImageJ software. Error bars show mean ± s.e.m; (n = 9 to 24 fields from n = 2 experiments performed in duplicate. Ordinary one-way ANOVA Tukey’s multiple comparison test ****P < 0.0001; *P < 0.05; NS, not significant (NS1 = 0.9508, NS2 = 0.4929, NS3 > 0.9999). i, Production of inflammatory markers in the cell supernatants. Error bars show mean ± s.e.m. of one representative experiment performed in triplicate. Ordinary one-way ANOVA Tukey’s multiple comparison test; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 NS, not significant (NS1 = 0.4623, NS2 = 0.3255).
