Extended Data Fig. 1: Expression profiles of bona fide and putative choline transporters.
From: Structural and molecular basis of choline uptake into the brain by FLVCR2
a, Expression profiles14,15 showing that Flvcr2 is enriched in brain endothelial cells in both human and mouse. Other putative and validated choline transporters are not specifically enriched at the BBB. Abbreviations on the horizontal axes indicate: astrocytes (astro), neuron, oligodendrocyte progenitor cells (OPC), immature oligodendrocytes (Olig1), mature oligodendrocytes (Olig2), microglia (MG), endothelial cells (EC), foetal astrocytes (FetalAC), maternal astrocytes (MatAC), and oligodendrocytes (Olig). Expression in endothelial cells is shown in orange; all other cell types are shown in green. Note the differences in vertical axis scale between transporters. b, Affinities of putative and validated choline transporters16. c, RNA sequencing expression profiles from multiple in vitro BBB models compared to acutely isolated brain endothelial cells (Brain EC): primary culture of brain EC, iHBEC, BEND3, HEK293, human iPSC-derived neurons, and HeLa cells (with and without Flvcr1 knockout), showing expression of putative and validated choline transporters in these cells. Flvcr1 expression level in Flvcr1 knockout HeLa cells is coloured grey to denote gene knockout by Crispr/Cas9. d, RT-qPCR performed with RNA extracted from iHBEC cells transfected with either scr siRNA (control) or Flvcr2 siRNA. Human Gapdh expression was used as the internal loading control. e, Fixed sections from Flvcr2HA/HA mice immuno-stained for HA (cyan) and vascular markers ICAM2 (luminal; pink) and COL IV (abluminal; dark blue). Projected Z-stack images (15 μm thick) and scale bars of 10 µm are shown. Each representative image was taken from three different biological samples and data analysed from four to five images from each brain. The right panel shows percentage co-localisation of FLVCR2-HA with ICAM2 and COL IV. f, Fixed human brain tissues were immuno-stained for FLVCR2 (cyan) using an FLVCR2 specific antibody and pan-endothelial markers CD34 (luminal; pink) and COL IV (abluminal; dark blue). Projected Z-stack images (15 μm thick) and scale bars of 10 µm are shown. Each representative image was taken from five different biological samples and data analysed from four to five images from each brain. The right panel shows percentage co-localisation of FLVCR2 with CD34 and COL IV. Graphical data represent mean ± s.e.m. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. Statistical tests used and exact P and n values are provided in Supplementary Tables 1 and 2.
