Fig 1: Blood–brain barrier cells are highly resistant to TRAIL treatment. (A) Surface expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 receptors in hCMEC/D3 and HCT116 cells were quantified by the QIFIKIT and flow cytometry. The mean ± SD of three independent experiments is shown. Statistical significance was tested by Tukey’s two-way ANOVA on log-transformed receptor values: **** = p < 0.0001, ** = p < 0.01. (B) hCMEC/D3 cells were treated with the indicated concentration of Fc-scTRAIL for 1, 2 or 6 h and then analysed for procaspase 8, cleaved caspase 8 (p18/p10), procaspase 3 and cleaved caspase 3 (p21/p19/p17) by western blotting. Representative images from two independent experiments are shown. (C) bEnd.3 cells were treated with the indicated concentration of Fc-scTRAIL for 1, 2 or 6 h and then analysed for procaspase 8, cleaved caspase 8 (p18/p10), procaspase 3 and cleaved caspase 3 (p21/p19/p17) by western blotting. Representative images from two independent experiments are shown. (D) hCMEC/D3 cells were treated with indicated construct for 24 h. Viable cells were determined by Annexin V-PI negativity using flow cytometry. Data are shown as mean ± SEM from three independent experiments. (E) bEnd.3 cells were treated with indicated construct for 24 h. Viable cells were determined by MTT assay. Data are shown as mean ± range from two independent experiments.