25HC dysregulates the function of human brain endothelial cells. (A) Permeability of fluorescein-conjugated dextran 70 kDa (FD70) in hCMEC/D3 cell monolayer pre-treated with different concentrations of 25HC (0-5 μM, 24 h). (B,C) hCMEC/D3 cell migration in a scratch assay; cells were pre-treated with different concentrations of 25HC (0-5 μM, 24 h before scratch). Representative images are shown for cells 0 and 24 h after scratch (B; 5 μM 25HC). Blue lines show the initial scratched area. Scale bars: 100 µM. Migration was quantified as relative wound density (C; n=10). (D) FD70 permeability was analysed in hCMEC/D3 cells treated with 25HC (5 μM, 24 h) and ATV (1 μM, 24 h). (E) Cell migration at 24 h post-scratch of hCMEC/D3 cells pre-treated with 25HC (5 μM, 24 h) before scratch and treated with ATV (1 µM, 24 h) after scratch. (F) FD70 permeability was analysed in hCMEC/D3 cells pre-treated with 25HC (5 μM, 14 h) and then supplemented with soluble cholesterol (80 µM, 1 h). (G) Cell migration at 24 h post-scratch of hCMEC/D3 cells pre-treated with 25HC (5 μM, 24 h) and then supplemented with cholesterol (80 µM, 2 h) before scratch. Data expressed as mean±s.d. ns, nonsignificant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; determined by randomised block one-way ANOVA with Dunnett's post-hoc test compared to 0 µM (A), matched measures two-way ANOVA with Dunnett's post-hoc test compared to 0 µM (C), or randomised block two-way ANOVA with Sidak's post-hoc test compared to control (D-G).
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