Fig. 4

Internalization of tPA CPECs is an active process, mediated by LRP1. A Representative photomicrographs (n = 5) of CPs harvested from mice intravenously injected with tPA⁵⁵⁵ (tPA⁵⁵⁵ in magenta, LRP1 in yellow, and TTR in white); panels show projection over the XZ axis (bottom) and over the YZ axis (right), scale bar 10 µm. Arrowheads point examples of tPA⁵⁵⁵ dots that can be found close to LRP1 staining in TTR positive cells. a: apical side; b: basal side. B Representative photomicrographs (n = 4) of cultured CPECs show LRP1 (yellow) and TTR (white), scale bar 40 µm. C Representative photomicrographs (n = 4) of CPECs exposed for 15 min to tPA⁵⁵⁵ (40 nM) alone or with RAP (500 nM), scale bar 40 µm. D Representative electrophoresis (representative of n = 4) and corresponding quantification (mean ± SEM, * significant difference (p < 0.05)), of protein extracts from monolayer of CPECs exposed for 15 min to tPA⁵⁵⁵ (40 nM) alone or with RAP (500 nM). E Representative western blot against LRP1 and corresponding quantification after transfection of cultured CPECs with nothing (Control), negative control (Scramble) or siRNA anti-LRP1 (siLRP1) (n = 4, mean ± SEM of LRP1 expressed as percentage of control). F Representative electrophoresis and corresponding quantification of protein extracts from monolayer of CPECs exposed for 15 min to tPA⁵⁵⁵ (40 nM) after transfection with control, scramble or siLRP1 (n = 4, mean ± SEM of LRP1 expressed as percentage of control). G–H mice were injected intravenously with 50 µg of tPA⁵⁵⁵ alone or together with 90 µg of RAP. 30 min later, PCs were freshly isolated for immunohistochemistry. G Representative confocal photomicrographs (n = 4) of a stack of CPs explants, with tPA⁵⁵⁵ (magenta), TTR (white) and DAPI (blue) staining. H Quantifications of tPA⁵⁵⁵ fluorescence intensity in CPs explants. Scale bar 50 µm and 10 µm in the magnified inserts