Fig. 4.

Establishment and characterization of Cas9 brain endothelial cell lines. a Representative confocal image acquired at the core of a blood-barrier organoid assembled with brain endothelial cells expressing Cas9 (orange). Cas9 brain endothelial cells localize only at the periphery of blood–brain barrier organoids. Nuclei labelled with DAPI (cyan). Scale bar, 100 μm. b Representative Western blot image showing the expression of Transferrin receptor, clathrin heavy chain or caveolin-1 in hCMEC/D3 brain endothelial cells expressing Cas9 and transduced with either scrambled gRNA or gRNA against the target gene. β actin expression is shown as a reference control gene. c Representative fluorescent images of hCMEC/D3 brain endothelial Cas9 or knockout cells after incubation with fluorescently labelled transferrin (yellow) for 30 min. Actin is labelled with phalloidin (grey) to visualize cell contours and nuclei are labelled with DAPI (cyan). Scale bars, 20 μm. d Quantification of transferrin internalization in hCMEC/D3 Cas9 or knockout cells. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles. Differences between the scrambled control and transferrin receptor or clathrin heavy-chain knockout cells were statistically significant (**p = 0.018) whereas the difference between the scrambled control and caveolin-1 knockout cells was not statistically significant (p = 0.418). Comparisons were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons of ~ 400 single cells per condition in n = 2 independent experiments