Fig 2: The TGF-ß signaling pathway determined the EMT level. (E–H) AECs were treated with 10 µg/mL SB431542 for 30 minutes. Western blots of Zo1 and vimentin in BEAS-2B (E) and HSAEpiC (F) cells. qRT-PCR analyses of Zo1 and vimentin in BEAS-2B (G) and HSAEpiC (H) cells. * p<0.05, ** p<0.01 compared with the control group; # p<0.05, ## p<0.01 compared with the CSE group SB: SB431542.

Fig 3: Effects of sh-SIRT1 on ZO-1 levels based on immunofluorescence staining of Tg-induced Caco-2 and IEC-6 cells. Data are presented as the mean ± SEM (n = 3). Statistically significant differences are indicated; *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from control group. #p < 0.05, significantly different from Tg group.

Fig 4: Autophagy inhibition protects against LPS-induced pulmonary endothelial injury. (A-D) MLVECs were treated with LPS (1,000 ng/mL) with or without autophagy inhibitor 3-MA (10 mM) for 24 hours. (A) Representative protein bands of VE-cadherin and ZO-1. The corresponding histograms were presented on the right of the bands. (B) Representative immunofluorescent staining images showed that 3-MA reversed LPS-induced loss of ZO-1 (green) expression in MLVECs. Nuclei were counterstained with DAPI (blue). Original magnification, ×200, scale bars correspond to 50 µm. (C) The intensity of ZO-1 immunofluorescent staining was shown in histograms. (D) Endothelial permeability was determined by FITC-dextran flux assay. (E-H) MLVECs were transfected with control siRNA or siRNA targeting ATG5. Forty-eight hours later, cells were treated with or without LPS (1,000 ng/mL) for another 24 hours. (E) Representative protein bands of VE-cadherin, ZO-1 and ATG5. The corresponding histograms were presented on the right of the bands. (F) Representative immunofluorescent staining images showed that ATG5 knockdown reversed LPS-induced loss of ZO-1 (green) expression in MLVECs. Nuclei were counterstained with DAPI (blue). Original magnification, ×200, scale bars correspond to 50 µm. (G) The intensity of ZO-1 immunofluorescent staining was shown in histograms. (H) Endothelial permeability was determined by FITC-dextran flux assay. Data are presented as the mean ± SEM (n=4). *, P<0.05; **, P<0.01 vs. control group; ##, P<0.01 vs. LPS group. ATG5, autophagy-related gene 5; DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate-carboxymethyl; LPS, lipopolysaccharide; MLVECs, mouse lung vascular endothelial cells; 3-MA, 3-methyladenine; RFU, relative fluorescence units; SEM, standard error of the mean; VE, vascular endothelial; ZO-1, zonula occludens-1.

Fig 5: Silencing AIM2 rescued BBB breakdown after OGD/R in vitro. A, Quantification of transendothelial permeability as determined by the TEER assay (n=5). B, Western blot analysis of ZO-1, occludin, and GAPDH after OGD/R. C, D, Quantification of the protein levels of ZO-1 and occludin normalized to those of GAPDH. E, Representative immunofluorescence images of DAPI (blue)/ZO-1 (red) colocalization after OGD/R. Scale bar: 20 µm. All data are presented as the mean ±SEM. ** p < 0.01 compared with the control group. # p < 0.05 and ## p < 0.01 compared with the OGD/R-control group