Figure 3.

Mechanisms of NMV internalization by hCMEC/D3 cells. (A) Co-incubation with EDTA, a metal ion chelator significantly reduced internalization. (B) NMV internalization was significantly reduced at 4 °C. (C) Pre-incubation with LPS (1 µg/mL) and TNF-α (10 ng/mL) did not affect internalization. (D) hCMEC/D3 cells were pre-incubated with pharmacological inhibitors genistein, wortmannin, MDC, EIPA, cytochalasin D, Dynasore, and also proteinase K and PKH-67 labeled NMV were added for 2 h. Data represent 3–5 independent experiments and are presented as the percentage of NMV uptake compared to the positive control with no inhibitors (mean ± SD, One-way ANOVA with Dunnett’s multiple comparison test * p < 0.05, *** p < 0.001, **** p < 0.0001).