Fig 2: Restoration of the vascular barrier coincided with reorganization of TJ complexes. A At the indicated times following IR injury, retinas were removed and proteins in whole retinal lysates examined by Western blotting with antibodies to occludin phosphorylated on serine 490 (pS490), total occludin protein, ZO-1, ZO-2, and claudin-5. A representative blot is shown. B Quantification of 4 replicate Western blot experiments. Occludin pS490 immunoreactivity was normalized to that of total occludin. ZO-1, ZO-2, and claudin-5 immunoreactivities were normalized to that of ß-actin. *p = 0.05, **p = 0.01, ***p = 0.001 by t test. C At various times following IR injury (2-day and 4-week samples shown), retinas were removed and immuno-probed with antibodies to occludin and ZO-1, followed by flat mounting and confocal microscopy. Arrows indicate discontinuities in TJ proteins at endothelial cell borders. D Histograms represent the evaluation of TJ continuity at endothelial cell borders using a blinded rank (1–5) scoring system. In the graph, green indicates completely continuous, yellow indicates 75% to 100% continuous, pink indicates 50% to 75% continuous, orange indicates 25% to 50% continuous, and red indicates 0% to 25% continuous border staining. For each retina, four images equidistant from the optic disc were averaged with n = 4 retinas for each condition. *p = 0.05, ****p = 0.0001 by t test

Fig 3: Pb exposure induced a low inflammatory response in the gut and gut dysbiosis in mice. (a) Western blot analysis and shading analysis of gut barrier markers, including E-cadherin, Claudin-3, Claudin-1, ZO-2, and ZO-1 in control and high-dose Pb exposure groups. (b) Representative images of hematoxylin and eosin-stained colon sections from control and high dose Pb exposure groups. (c) Representative images of HE stained jejunum sections from control and high dose Pb exposure groups. (d) Shannon diversity index of gut microbiota in the three groups. (e) PCOA analysis of gut microbiota in the three groups. *P < 0.05.**P < 0.01.

Fig 4: Reduced TER in CST3 B/B iPS-RPE cells. (A) TER of WT and gene-edited iPS-RPE cells seeded on transwell inserts was monitored over 8 weeks. (B) Conditioned media from the upper and lower wells of transwell inserts, corresponding to apical and basolateral secretion, were analyzed by Western blot for cystatin C and PEDF secretion. A, apical secretion; B, basolateral secretion. (C) Expression of tight junction proteins ZO1, ZO2, and claudin 3 was confirmed by Western blot. Images shown are representative of three individual experiments. (D) Densitometric analysis of tight junction protein content from whole-cell lysates shown in panel C. n = 3; *, P = 0.05.

Fig 5: Gene expression levels of Claudin-1, ZO-1 and ZO-2 mRNAs in BA and BA-Al NPs-treated Caco-2 cells. Caco-2 cells treated with DMEM medium were used as a blank control * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (a); Representative western blot of Claudin-1, ZO-1 and ZO-2 proteins in BA and BA-Al NPs- treated Caco-2 cells (b); Quantification of Claudin-1, ZO-1 and ZO-2 proteins using Image J. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 (c); Immunofluorescence staining of TJ protein ZO-1 in BA and BA-Al NPs-treated Caco-2 cells (d); Molecular docking analysis of interaction between BA and 2h3m (crystal structure of ZO-1 PDZ1) (e); Schematic illustration showing the mechanism and consequence of BA-Al NPs mediated TJs modulating between intestinal epithelial cells (f)