Fig 2: Effect of NCZ on P-gp-Mediated hAß42 transport. a Representative confocal images of isolated brain capillaries showing accumulation of fluorescein-hAß42 in capillary lumens after a 1 hour incubation (steady state; 5 µM fluorescein-hAß42). b Data after digital image analysis using ImageJ. Specific fluorescein-hAß42 fluorescence is the difference between total luminal fluorescein-hAß42 fluorescence and fluorescein-hAß42 fluorescence in the presence of the P-gp-specific inhibitor PSC833 (5 µM). Statistics: Data per group are given as mean ± S.E.M. for 10 capillaries from one preparation (pooled tissue: WT (10 mice), hAPP (15 mice), hAPP-NCZ (15 mice), hAPP-NCZ/CSA (15 mice)). Shown are arbitrary fluorescence units (scale 0–255). ***, significantly lower than control, p < 0.001

Fig 3: Specialized EPDC’s BBB characteristics.(A) Quantitative RT-PCR expression analysis of specific BBB genes at day 14 of culture, in EPDCs alone (purple), with astrocytes (blue) and hCMEC-D3 (green) (qRT-PCR were performed on EPDCs RNA isolated from 6 BBB differentiation independent experiences, each individual experience was performed in triplicate). (B) Western blot of P-gp, GLUT-1 and OCCL in EPDCs alone (purple) or with astrocytes (blue) at day 14. Densiometric quantification was performed for each immunoblot using ImageJ software. (C–L) VE-CAD, ZO1, CL3, CL5 and OCCL immunofluorescence staining in EPDCs alone (C–G) or with astrocytes (H–L). Arrows show continuous junctions. (M) Permeability for LY (0.457 kDa) and Dextran-FITC (4 and 70 kDa) in EPDCs alone (purple) or with astrocytes (blue) and in hCMEC/D3 (green) (EPDC permeability to dextran-FITC molecules was performed once in triplicate). (N) Accumulation of Calcein into EPDCs alone or with astrocytes, in the presence (hatched area) or absence of Verapamil (**p<0.001; a.u: arbitrary units). Scale bar: SEM.

Fig 4: PYR41 prevents reduction of P-gp protein expression and transport activity in human amyloid precursor protein (hAPP) mice. (A) Western blot showing that PYR41 treatment restored P-gp protein expression levels in hAPP mice compared to vehicle-treated wild type (WT) mice; ß-actin was used as protein loading control. (B) Representative confocal images of brain capillaries isolated from WT, hAPP, hAPP-PYR41 and hAPP-PYR41/CSA mice after a 1 h incubation (steady state) with the fluorescent, P-gp-specific substrate NBD-CSA (2 µM). (C) Data from ImageJ analysis of confocal images in (B) show that luminal NBD-CSA fluorescence was significantly reduced in brain capillaries from hAPP and hAPP-PYR41/CSA mice relative to brain capillaries from WT and hAPP-PYR41 mice. Specific NBD-CSA fluorescence is the difference between total luminal fluorescence and fluorescence in the presence of the P-gp-specific inhibitor PSC833, representing transport activity that is specific to P-gp. Data are mean ± SEM (n = 10 capillaries per treatment group); arbitrary fluorescence units (scale 0–255). Statistics: ***p < 0.001 analysis of variance (ANOVA), significantly lower than WT control group.

Fig 5: ZIP1+ fibroblasts promote chemoresistance of lung cancer cells in vitro and in vivo.a Experimental design (left) and results of DNA damage (?-H2AX expression) in LLC-GFP-luc cells co-cultured with Zip1+/+ or Zip1-/- MEFs with DOX (3 µM) treatment. Mean ± SEM, n = 4. Two-tailed t-test. b DOX accumulation in LLC-GFP-luc tumour cells co-cultured with Zip1+/+ or Zip1-/- MEFs with DOX (3 µM) treatment. Mean ± SEM, n = 4. Two-tailed t-test. c ABCB1 expression in LLC-GFP-luc tumour cells co-cultured with Zip1+/+ or Zip1-/- MEFs for 24 h. Mean ± SEM. Ctl, Zip1+/+: n = 3; Zip1-/-: n = 6. Two-tailed t-test. d DOX accumulation in LLC-GFP-luc tumour cells 6 h after DOX injection in tumour-bearing Zip1+/+, Zip1+/- and Zip1-/- mice. DOX (10 mg/kg) was intravenously injected into the mice at day 10 post-transplantation. Mean ± SEM. Zip1+/+: n = 4; Zip1+/-, Zip1-/-: n = 5. One-tailed Kruskal–Wallis test. e ABCB1 expression in LLC-GFP-luc cells transplanted into Zip1+/+ and Zip1-/- mice at day 10 post-transplantation. Mean ± SEM, n = 5. Two-tailed t-test. f Tumour growth curves for Zip1+/+ and Zip1-/- mice treated with DOX or PBS. Arrows, DOX injection. Mean ± SEM. P values are for indicated time points. Zip1+/+ PBS (n = 11), Zip1-/- PBS (n = 12), Zip1+/+ DOX (n = 8), Zip1-/- MEFs DOX (n = 10). g Tumour growth curves for co-injection of Zip1+/+ or Zip1-/- MEFs with LLC-GFP-luc cells, with DOX or PBS treatment. Arrows indicate DOX injection. Zip1+/+, Zip1-/- MEFs PBS, Zip1-/- MEFs DOX: n = 9; Zip1+/+ MEFs DOX: n = 6. Mean ± SEM. h Tumour growth curves for mice inoculated with LLC cells and mCAFs with a tet-off system-controlled expression of Zip1 (TET-Zip1). Mice were administered PTX, with or without doxycycline (Dc) treatment. Mean ± SEM. Arrow, PTX (10 mg/kg) treatment. TET-mock, TET-Zip1 PBS: n = 6; TET-Zip1 PTX, TET-Zip1 PTX + Dc: n = 5. Two-way ANOVA test for tumour growth curve comparison. Source data are provided as a Source Data file (a–h).