Fig. 5. Roles of 5HT2A receptor (5HT2AR) and clathrin-mediated endocytosis (CME) in mediating VLP uptake into BBB cells. hCMEC/D3 cells (a) and human iPSC-derived astrocytes (b) were pre-treated by 5HT2AR antibody of 0, 0.185 and 1.85 μg mL⁻¹ for 1 h and then exposed to 31.5 μg mL⁻¹ VLPs for 24 h. The VLP uptake was assessed based on dependence of antibody treatment or cell type. For inhibition of CME, hCMEC/D3 cells were pre-treated by either chlorpromazine (CPZ) at 0, 1 or 100 μM for 1 h (c), or by Pitstop2 inhibitor and Pitstop2 negative control, both at 30 μM for 0.5 h (d). VLPs were subsequently applied for 1.5 h before cellular uptake was evaluated by HCA. Bar graphs were generated as described in the Experimental section. Error bars represent means ± SD from triplicate wells. One-way ANOVA analysis was applied to determine statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and no significance (ns) at p ≥ 0.05).