Additional file 1 of The choroid plexus: a door between the blood and the brain for tissue-type plasminogen activator
Creators: name: Zuba, Vincent
name: Furon, Jonathane
name: Bellemain-Sagnard, Mathys
name: Martinez de Lizarrondo, Sara
name: Lebouvier, Laurent
name: Rubio, Marina
name: Hommet, Yannick
name: Gauberti, Maxime
name: Vivien, Denis
name: Ali, Carine
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1. In contrast to tPA coupled to Alexa, Alexa alone is not taken-up by CPECs in vivo. A, Experimental design: mice were injected intravenously with of tPA555 and Alexa488 (50µg each), brains were harvested 30min later for immunohistochemistry. B, Representative confocal photomicrographs (n=4) of CPs sections with tPA555 (magenta), Alexa488 (green) and DAPI (blue) staining; scale bar 50µm (upper panels) and 10µm in the magnified inserts (lower panels). Figure S2. CPECs internalized tPA by clathrin-coated endocytosis vesicles. A, Confocal photomicrographs (representative of n=6 slices) of CPs sections with tPA555 (left bottom, magenta), TTR (left top, white) and AP2 (right top, green) staining; scale bar 50µm and 10µm in the magnified inserts. B, Image analysis of confocal photomicrographs using the Imaris Microscopy Image Analysis Software to study the colocalization between tPA555 and AP2 staining (yellow, right). tPA endocytosis (white arrow); scale bar 10µm. Figure S3. Existence of an efflux pathway for tPA. A, Experimental design: mice received an intra-cerebroventricular injection of tPA555 (1µl at 0.9mg/ml), and brains were harvested 10, 30 or 60min later for immunohistochemistry. B, Representative confocal photomicrographs (n=4) of CPs sections with tPA555 (magenta), LRP1 (green), TTR (white) and DAPI (blue) staining; scale bar 50µm and 10µm in the magnified inserts. Figure S4. Evolution of tPA555 levels in the blood and CSF. A, Experimental design illustrating that mice were injected intravenously with 50µg of tPA555 and BSA488, CSF, blood and brains were harvested 15, 30, or 45min later for protein analysis by electrophoresis (2µl of CSF and 1 µl of plasma were resolved for each animal). B, Representative electrophoreses and C, corresponding quantification for tPA555 levels in the plasma and in the CSF (mean ±SEM, n=5). ** significant difference (p&lt;0.005). Figure 5: tPA555 is rapidly taken-up by CPECs after intra-carotid injection. A, Experimental design: mice were injected in the carotid with 50µg of tPA555, brains were harvested 5min later, and PCs freshly isolated for immunohistochemistry. B, C, D: Representative confocal photomicrographs (n=5) of a stack of CPs explants with tPA555 (magenta) and DAPI staining (blue), focus on PCs vasculature (B), on PCs stroma (C) and on CPECs (D). Scale bar 50µm and 10µm in the magnified inserts. Figure S6. Confocal photomicrograph shows tPA555 (magenta) and BSA488 (green) extravasation out of vessels (CD31 staining in white), in the Median Eminence, a barrier free zone; DAPI staining (blue), scale bar 40µm