40-2300

antibody from Invitrogen Antibodies

Targeting: TJP1 DKFZp686M05161, MGC133289, ZO-1

Provider product page for 40-2300

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Antibody data

Antibody Data
Antigen structure
References [92]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [2]
Immunohistochemistry [3]
Other assay [76]

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Product number: 40-2300 - Provider product page
Provider: Invitrogen Antibodies
Product name: ZO-1 Polyclonal Antibody (ZMD.437)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody reacts with the ZO-1 protein. Based on sequence homology, reactivity with ZO-2 or ZO-3 is not expected. 40-2300 has been successfully used in immunofluorescence and immunohistochemistry. However, for western blotting we recommend the use of monoclonal anti-ZO-1 antibody (Cat. No. 339100), or other polyclonal ZO-1 antibodies (Cat. Nos. 617300 or 402200).
Antibody clone number: ZMD.437
Concentration: 0.25 mg/mL

TJP1 protein structure - 40-2300 shown in red.

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Experimental details: Figure 3 ZO-1 and E-cadherin localization are unaffected by LKB1 RNAi in MDCK cell cysts. Representative images of ZO-1 (red) and E-cadherin (green) immunofluorescence in control MDCK cell cysts and in cysts with LKB1 RNAi are shown. Sections were taken through the midpoint of the cyst to show the hollow lumen as well as the apical and lateral surfaces of cells at the widest part of the cyst structure. Bars, 10 um.

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Experimental details: Figure 1. E-cad deletion induced loss of simple epithelial architecture. (A) E-cad deletion was induced in half of Cre-ER;E-cad fl/fl organoids with tamoxifen. (B) Control, E-cad + organoids (-Tam) formed cysts. (C) E-cad - organoids (+Tam) failed to form cysts (28/42 movies across three biological replicates) or transiently established and then lost lumens (14/42 movies). (D) E-cad deletion blocked cyst formation. n , total number of organoids; r , number of biological replicates. Error bars indicate SD. ***, P = 0.0004, two-tailed Student's t test with equal variance. (E) Control organoids formed cysts with enrichment of E-cad and ZO-1 along apicolateral membranes (E'). (F) E-cad - organoids were multilayered, lacked E-cad immunoreactivity, and displayed abnormal ZO-1 localization. Arrow indicates rare E-cad + cells. (G) By Western blot, E-cad deletion (+Tam) resulted in complete loss of E-cad protein and significant reductions in alphaE-catenin and beta-catenin (see also Fig. S1, A and B ). Whole cell lysate samples were loaded for equal protein based on BCA analysis. (H) The Cre biosensor mT/mG was used to observe E-cad - cell behaviors by confocal microscopy ( Video 1 ). Cre + , E-cad - cells (green) changed shape, from columnar to round, before shifting apically (H' and H'', arrowheads). Gamma adjustments were performed in E and F to improve image clarity. Bars: (B and C) 20 um; (E, F, and H) 10 um.

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Experimental details: Figure 5 Overexpression of Prox1 results in changes in junctional proteins in vivo and in vitro . (A) RT-PCR analysis was performed on yolk sacs derived from E13.5 embryos to targets such as ZO-1, Occludin and JAM-1. (B) Similarly, in vitro analysis shows that overexpresion of Prox1 in vascular endothelial cells decreases junctional protein levels. (C) Bright field microscopy of VECs transfected with Prox1 (10x mag) (D) Vascular endothelial cells that overexpress Prox1 display an increase in permeability measured by FITC-albumin transit in a Boyden chamber assay. Figure is representative of three experiments with all samples run in triplicate. Error bars represent standard deviation of the mean. P < 0.05.

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Experimental details: Fig. 5 Effects of IR injury on ZO-1 expression in the outer medulla. Representative micrographs of ZO-1 localization in the outer medulla in control ( n = 5) and IR ( n = 5) kidneys. In control kidneys ( a , b ), ZO-1 ( brown ), immunoreactivity was observed in the proximal tubule ( PT ) as well as in the collecting duct ( CD ). In ischemic kidneys ( c , d ), ZO-1 localization was distorted and abnormally localized in the cytoplasm ( arrowheads , inset). Occasionally, a combination of both delocalization and upregulation of ZO-1 expression was observed in some cells ( arrows ) in the PT and CD

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Experimental details: Figure 4 MUC4 contributes to a altered phenotype. ( A ) The control or MUC4 knockdown cells were seeded in 2% Matrigel on top of a 100% Matrigel layer, and fed with media every 3 days. After 7 days, acini-like structures were photographed under a phase-contrast microscope. The acini-like structures (examples shown in the boxes) that were regular (smooth and spherical shape) or irregular (irregular outline, multi-lobular) were counted and plotted as a percentage of the total count (p = 0.0005 for regular and p = 0.002 for irregular). A minimum of 120 structures was counted for each of control cells or MUC4 knockdown cells. Reduced irregular outline, multi-lobular and increased smooth and spherical shape colonies were found in MUC4 knock down cells when compared with control cells. ( B ) Structures were stained with the anti-ZO-1 antibody. 4,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Optical sections (0.7-0.9 um) were captured using a laser scanning confocal microscope. The images presented here are the central planes of the acini. Bar = 20 um.

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Experimental details: Figure 6. The ratio of lumen coalescence to cell division times controls the monolumen or multilumen phenotypes. (A) We treated cysts with aPKC-PSi to induce polarity disruption and spatially disordered spindle positioning. Cells were seeded in growth medium supplemented with 50 uM aPKC-PSi, and fixed 3 d later. Confocal scans for cysts treated with aPKC-PSi, which lack control over mitotic spindle positioning; stained for beta-catenin (green), labeling cell membranes; and ZO-1 (white) and PCX/gp135 (red), labeling luminal interfaces. Notice the multilumen morphology. Time from seeding: 3 d. Bar, 10 um. (B) 3D reconstructions of the cyst in A from the full set of confocal sections. Several disconnected lumens are visible in the cross-section (right). Bar, 10 um. (C) Mean number of luminal volumes and fraction of cysts with single lumen/multiple lumen phenotypes (inset), obtained from numerical simulations as a function of the tightness of the control over orientation of the cleavage plane. ph = 0deg, mitotic planes always orthogonal to the luminal surface. ph = 90deg, division planes chosen isotropically in three dimensions. Note that even for ph = 0deg, it is still possible to get more than one lumen. Simulated cysts have 32 cells. Error bars indicate standard deviations. (D) Numerical simulation of a multiple-lumen phenotype. The cleavage plane has been chosen at random, isotropically in 3D space. Equivalent time from seeding is around 5 d. (E) Mean fraction of cysts with s

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Experimental details: Figure 3 Perivascular glioma cell co-option causes a breakdown of the blood-brain barrier The tracer, Evans blue, permeates into the brain parenchyma in tumor-bearing ( n =3 animals) (bottom), but not in sham mice 3 weeks post surgery ( n =3 animals) (top), Scale 5 mm. (a) . Immunofluorescence of CD31 and intravenously injected tracers, tetramethylrhodamine-albumin (TRITC-alb, white) or -cadaverine (TRITC-cad, white), and implanted eGFP-expressing human glioma cells ( D54 ) allow analysis of tracer leakage in relation to tumor burden. The large MW 70 kDa TRITC-albumin (b) , as well as the small MW 950 Da TRITC-cadaverine (d) can be found outside the vasculature (arrows) (b-f , Suppl. Fig.6 ) . Note that leaked TRITC-cadaverine is taken up by nearby neurons (arrows) (d,e) . No extravasation was seen in the absence of tumor cells (c,f) . To assess extravasation of the tracer Cascade blue (MW 10 kDa), Aldh1l1-eGFP-scid (eGFP) immunodeficient mice, which were previously implanted with TdTomato-expressing human glioma cells ( D54 ), were retro-orbitally injected with Alexa Fluor 633 hydrazide dye (633 hyd) and Cascade blue (g,h) . Accumulation of Cascade blue occurs in the brain's parenchyma indicating breakdown of the blood-brain barrier (BBB), where perivascular astrocytes have been displaced by glioma cell co-option (g) . Vessels lacking tumor cells do not show extravasation of the dye (h) . Immunohistochemistry for the TJ proteins zonula occludens-1 (ZO-1) (i) and claudin

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Experimental details: Fig. 4 Endothelial cells were infected for 2 h with S. aureus (MOI 10:1) or left uninfected. As indicated, cells were pretreated for 20 min with amitriptyline (Ami) (20 muM), Tiron (10 mM), or NAC (10 mM) before infection with S. aureus . Immunofluorescence stainings were performed with antibodies against ZO1, ZO2, occludin, or E-cadherin for determination of the degradation of these TJ proteins. The presented pictures are representative of the results of at least three independent experiments. Scale bar is 25 mum

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Experimental details: Fig 1 Properties of cultured mLSECs. (A) Phase contrast microscopy of p19 ARF-/- mLSECs at passage 5. Inset show cells at higher magnification. (B) Proliferation kinetics by cell density analysis (upper panel) and proliferation kinetics by determination of cumulative cell numbers (lower panel) of mLSECs at early (p5) and late passages (p20). (C) Immunofluorescence analysis showing endothelial junction and intermediate filament proteins expressed in early and late passaged mLSECs. *p

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Experimental details: Fig 4 Tight junction formation by neonatal RBEC under static and flow conditions as indicated by immunofluorescence staining of ZO-1. RBEC cultured for 5 days under static conditions were stained with ZO-1 (red) and Hoechst 33342 (blue) (A). RBEC cultured for 5 days under flow of RBEC medium in B 3 C were stained with ZO-1 (red) and Hoechst 33342 (blue) (B). RBEC cultured under flow of ACM for 5 days in B 3 C were stained with ZO-1 (green) and Hoechst 33342 (blue) (C). Bright field image of B 3 C showing RBEC in the vascular channels after 5 days of co-culture with rat astrocytes in the tissue compartment of B 3 C (D). Immunofluorescence staining of RBEC in vascular channel for ZO-1 (green) and astrocytes in tissue compartment for GFAP (red) (E). Magnified view of interface with pores from panel E showing staining of cells inside the pores, ZO-1 (green), GFAP (red) and nuclei (blue) (F).

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Experimental details: Fig 8 Neonatal RBEC exhibit distinct barrier structure and function compared to adult RBEC in B 3 C. Neonatal RBEC alone (A) and neonatal RBEC + ACM (B) exhibit distinctly weaker and less organized ZO-1 staining (arrows) compared to adult RBEC alone (C) and adult RBEC + ACM (D). In the presence of ACM, neonatal RBEC exhibit discontinuous bands of ZO-1 staining (B) compared to neonatal RBEC in the absence of ACM where ZO-1 staining is discontinuous and granular (A). Presence of ACM has a significantly larger impact on both permeability (E) and electrical resistance (F) in neonatal RBEC compared to adult RBEC. Inset panels show higher magnification of white squared regions. (** indicates significant difference p

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Experimental details: Figure 4 Abating MICAL2 in 786-O kidney cancer cells in vitro induces MET ( A ), Morphological analysis in light-transmitted microscopy. Scale bar: 100 mum. ( B ), F-actin staining. IF of: E-cadherin, ZO-1, catenin-beta. In KD2 and KD14 cells, these three markers relocated at cell perimeters (as in epithelial cells), rather than being diffusedly cytoplasmic. IF of vimentin with Vim13.2 antibody. In MIC2-KD cells it decorated short and disorganized filaments instead of long meshes observed in control cells. Scale bar of all IF images: 20 mum. Inset magnification: 3x. ( C ), Vimentin epitope recognized by V9 antibody was undetectable in KD2 and KD14 cells. ( D ), Agarose gel run of SNAI1, SNAI2, 18s5 RT-PCR products. ( E ), KD14 cells transfected with HA-MICAL2 cDNA. The elongated phenotype showed dose-dependency on HA-MICAL2 expression. One-way Anova test and Tukey's Multiple Comparison post-hoc test. In all graph bars, horizontal lines denote mean and SEM. N.s: non significant. * p

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Experimental details: Figure 5 miR-143 regulated the permeability of endothelial cells by targeting PUMA. (a,b) The transduction of cells with the PUMA siRNA lentivirus decreased the PUMA expression and the expression of tight junction proteins (claudin-5, occludin, and ZO-1) in HBMECs. (c,d) The transduction of HBMECs with the PUMA OE lentivirus increased the expression of PUMA and tight junction proteins (claudin-5, occludin, and ZO-1) in HBMECs. (e) The transduction of cells with miR-143 failed to decrease the level of tight junction proteins in the cells co-transduced with the PUMA OE lentivirus, as determined by western blot analysis. (f) The transduction of cells with the PUMA siRNA lentivirus significantly inhibited the anti-miR-143-induced increase in the expression of tight junction proteins, as determined by western blot analysis. All data are presented as the mean +- SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the miR-Con/Vector group or the anti-miR-Con/siRNA-Con group; + p < 0.05 and ## p < 0.01 vs. the miR-143/Vector group or the anti-miR-143/siRNA-Con group using one-way ANOVA followed by the Holm-Sidak test. Meth, methamphetamine.

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Experimental details: Figure 4 Acini disorganization in HIF2alpha-overexpressing mice. No changes in the expression or localization of laminin ( A , B ), beta-catenin ( C , D ), and E-cadherin ( E - L ) between acini of two-week-old Pdx1-Cre;HIF2dPA and control mice. Carboxypeptidase A1 (CPA1) is localized close to the lumen of control acini ( E ). CPA1 does not show a clear apical localization in Pdx1-Cre;HIF2dPA acinar cells ( F ). Intracellular accumulation of mucin-1 ( H ), and PKCepsilon ( J ), in Pdx1-Cre;HIF2dPA acini while markers are normally localized at the apical region in control acini ( G , I ). Zonula occludens 1 (ZO-1) immunofluorescence show lumen dilation in Pdx1-Cre;HIF2dPA ( L ) acini compared to control acini ( K ). Nuclei are stained with DAPI (blue). Individual acini are outlined in yellow. Scale bars = 20 um.

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Experimental details: Fig. 1 Occludin and ZO-1 expression in control kidney. Representative of occludin ( a , b ), ZO-1( c - e ), and occludin-AQP1 confocal ( f - h ) immunostaining images obtained from sham-operated control kidneys. Occludin ( brown ) immunoreactivity was observed as dot-and-line patterns at the apical end of the lateral membrane in tubular epithelial cells in the cortex ( a ) and outer medulla ( b ). Occludin was expressed primarily in the distal tubule ( DT ) including the thick ascending limb ( TAL ). No occludin immunolabel was detected in glomeruli ( G ), proximal tubule ( PT ), or blood vessels, including the vascular bundles ( VB ). ZO-1 ( brown ) immunoreactivity was observed in all tubular segments, including the proximal tubule ( PT ) and distal tubule ( DT ) ( c - e ). ZO-1 labeling also appeared in the glomerular parietal epithelial cells ( arrows ) and in endothelial cells ( arrowheads ) of all blood vessels including the glomerular capillary, peritubular capillary, arcuate artery ( AA ), and vascular bundle ( VB ). To identify the thin limbs of the loop of Henle, confocal immunofluorescence staining for occludin ( red ) and AQP1 ( green ) were performed ( f - h ). Occasionally, transition points ( white arrows ) from the AQP1-positive descending thin limb ( DTL ) to AQP1-negative ascending thin limb ( ATL ) were observed and occludin immunoreactivity appeared in both thin DTL and ATL segments ( g , h ). Results are representative of findings in the five control rat

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Experimental details: Figure 1 Cell organization defects occur in the intestinal epithelium of CTE patients. ( a , b ) Histological analysis of hematoxylin-eosin stained paraffin sections of duodenal biopsies from control ( a ) and CTE patients ( b ). Scale bars, 10 mum. For each condition, a corresponding scheme of the epithelial organization is presented. CTE intestinal epithelium displays tufts (black arrowheads). ( c , d ) Confocal microscopy analysis of EpCAM distribution in duodenal control biopsy ( c ) or in Caco2 cells ( d ). Intestinal epithelium baseline is demarcated by dotted white line ( c ). Scale bars, upper panel 50 mum ( c ), lower panel 10 mum ( c ) and 10 mum ( d ). ( e ), Confocal microscopy analyses of Na + /K + -ATPase, E-cadherin, claudin-7 and ZO-1 distribution in control or CTE biopsies. N (Control) =6 biopsies, N (CTE) =6 biopsies. Scale bars, 10 mum. ( f ) Transmission electron microscopy (TEM) ultrastructural analysis of enterocyte apical membranes in control ( a ) and CTE ( b , c ) biopsies, showing actin rootlets ( black arrowheads ). Scale bars, 5 mum. N (Control) =3 biopsies, N (CTE) =3 biopsies. ( g ) Statistical analysis of microvillus density in control and CTE enterocytes. t test, * P

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Experimental details: Figure 2 EpCAM silencing leads to unusual tight junction positioning defects at tricellular junctions. ( a ) Western blot analysis of the EpCAM expression level in control ( Caco2 shNT ) or EpCAM-deprived ( Caco2 shEpCAM #1 and Caco2 shEpCAM #2 ) clones. GAPDH was used as loading control. ( b , c ) Confocal microscopy analysis of E-cadherin ( b ) and Scribble ( c ) on the apical or lateral sides in control and EpCAM-depleted cells. Scale bars, 5 mum. Nuclei were detected with Hoechst 33342 staining (blue). ( d , e ) 3D-SIM microscopy analysis of E-cadherin localization in control ( d ) and EpCAM-deprived ( e ) cells. Scale bars, 2 mum. ( f ) Cell shape analysis after E-cadherin immunostaining in control or EpCAM-deprived cells. Scale bars, 5 mum. ( g ), Statistical analysis of polygonal shape in control or EpCAM-deprived cells. Three independent experiments were carried out. N (Caco2shNT)=517 cells, N (Caco2shEpCAM#1)=550 cells, N (Caco2shEpCAM#2)=427 cells. Caco2 shNT cells with two cell edges=0%, three cell edges=0.455+-0.787%, four cell edges=8.673+-3.406%, five cell edges =48.154+-11.123%, six cell edges=35.652+-8.888%, more than six cell edges=7.067+-3.851 cells%. Caco2 shEpCAM#1 cells with two cell edges=1.822+-0.733 cells%, three cell edges=22.031+-5.906%, four cell edges=46.061+-3.851%, five cell edges=24.728+-4.344%, six cell edges=5.027+-2.210%, more than six cell edges=0.330+-0.572%. Caco2 shEpCAM#2 cells with two cell edges=0.966+-0.848%, three cell edges=12.542+-

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Experimental details: Figure 3 Representative photographs of claudin-2, claudin-3, claudin-4, and zonula occludens-1 immunostained in longitudinal jejunal sections of the three experimental groups. Claudin-2 immunoreaction in the sham-operated group (a) was low and was mainly found in the cytoplasm of the intestinal epithelial cells. Claudin-2 expression in the groups exposed to ischemia-reperfusion was substantially higher and was principally found in the lateral membrane of the epithelial cells (b, c). The sham-operated group presented strong membrane claudin-3 immunostaining (d), which was remarkably decreased in the ischemia-reperfusion- (IR-) group (e), but not in the postconditioned- (PC-) group (f). Minimal claudin-4 immunostaining was observed in the jejunal samples of the sham-operated animals (g). The two groups challenged with 60 minutes of mesenteric ischemia and 6 hours of reperfusion (h, i) showed high membrane claudin-4 expression, which was limited to the tips of the villi. Zonula occludens-1 was strongly immunostained in the jejunal samples of the sham-operated animals (j). Following small intestinal ischemia-reperfusion, intestinal epithelial cell with zonula occludens-1 membrane immunostaining notably decreased in the IR-group (k); in the PC-group minimal loss of zonula occludens-1 membrane immunostaining was detected (l). Each photograph was taken with the same magnification. Scale bar: 20 mu m.

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Experimental details: Immunolabelling experiments of Vitamin D receptor (VDR) and tight junction proteins in normal and psoriatic skin. Photomicrographs of skin specimens labelled by double immunofluorescence (a-f). Immunofluorescent staining of VDR (red) (a,d) and claudin-1 (green) (b,e) in normal (a-c) and psoriatic (d-f) skin showed a larger number of claudin-1- and VDR-positive cells (arrowheads) in normal compared to psoriatic skin. VDR and zonulin-1/occludin immunohistochemistry (g-l). Immunohistochemical staining in serial section from normal (g-i) and psoriatic (j-l) skin showed an altered expression of zonulin-1/occludin localization in psoriasis compared to that in normal skin. The number of clearly positive cells (arrowheads) is slightly larger in normal compared to psoriatic skin for both zonulin-1 (h,k) and occludin (i,l). However, the distribution of zonulin- 1/occludin involved a thicker region of the upper layers of psoriatic epidermis compared to normal one (double-headed arrows). Zonulin-1 and occludin decorated plasma membrane of keratinocytes with a discontinuous pattern in psoriatic compared to normal skin (arrowheads, high power fields, h,i,k,l). VDR-positive cells were mainly localized in the same areas in which zonulin-1- and occludin-positive cells were found (arrowheads, g,j, standard and high power fields). Original magnification, x400 (a-c), x200 (d-l); high power fields (x400) calibration bar: 25 mum (a-f), 50 mum (g-l). VDR was stained in red using an ALEXA 568-labe

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Experimental details: Figure 1 Effects of catecholamines and inflammatory mediators under normoxia conditions on cEND cells. Cells were grown to confluence with subsequent differentiation. Catecholamines (CAT) or inflammatory mediators (INF) were applied for 24 h under normoxia conditions (24 h NORMOX). (A,B) Immunofluorescence staining of tight junction proteins claudin-5 (green) and ZO-1 (red) as markers of morphological changes of the endothelial cell monolayer. DAPI (blue) was used for staining of nuclei. Magnification 400 times, scale bar 20 mum. (C) Cell number after CAT and INF treatment normalized to control. (D) CAT induced changes of ZO-1 protein expression in cEND cells investigated by Western blot. Data is presented as the means (+- SEM) of n = 5 independent experiments. Altered protein expression was normalized to beta-actin. ZO-1 protein level changes are expressed as fold over untreated control, which was set 1. * P < 0.05, ** P < 0.01, *** P < 0.001.

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Experimental details: Figure 2 Loss of morphological integrity of cEND cells exposed to elevated catecholamine levels and inflammatory mediators. Immunofluorescence staining of tight junction proteins claudin-5 (green) and ZO-1 (red) as markers of morphological changes of the endothelial cell monolayer. DAPI (blue) was used for staining nuclei. Cells were grown on cover slips to confluence. After differentiation cEND cells were treated with combination of catecholamines and inflammatory mediators (stress factors). Stress factors were administered under different incubation conditions. Magnification 400 times, scale bar 20 mum. (A) Stress factor application for 4h under normoxia conditions (4 h NORMOX). (B) Stress factor application for 24 h under normoxia conditions (24h NORMOX). (C) . Stress factor application for 4 h under oxygen glucose deprivation conditions (OGD). (D) Stress factor application for 4 h under OGD conditions with 20 h of subsequent reoxygenation under normoxia conditions (REOX). (E) Cell number in treatments shown in (A-D) normalized to control. *** P < 0.001, **** P < 0.0001.

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Experimental details: Figure 3 Protein expression in cEND cells is changed by elevated catecholamine levels and inflammatory mediators under oxygen glucose deprivation conditions. Cells were grown to confluence with subsequent differentiation. Stress factors were applied under different incubation conditions. Protein levels were examined by Western blot. Changed protein expression was normalized to beta-actin. Changes of protein levels determined by densitometric analysis are expressed as fold over untreated control, which was set as 1. (A) VE-cadherin protein expression after stress factor administration under oxygen glucose deprivation conditions (OGD) and subsequent reoxygenation (REOX) in comparison with 4 h of normoxia (4 h NORMOX). Values are the means (+- SEM) of n = 8 independent experiments. (B) Occludin and ZO-1 protein levels changed by stress factors after reoxygenation conditions (REOX) compared to 24 h of normoxia conditions (24 h NORMOX). Values are the means (+- SEM) of n = 10 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

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Experimental details: FIGURE 2 Cell seeding procedures and formation of a human brainmicrovessel using TY10 cells. (A) Cell seeding, 1st method. Representation of the steps used to increase the cell concentration before injection into the chip. Cells are allowed to settle by gravity (left and central panels) in the cell seeder and then injected as a bolus into the hollow lumen. Prior to injection, the chamber is capped with a plugged pipette tip (central panel). (B) Cell seeding, 2nd method. Representation of the steps used to inject cells into the chip in a more controlled and uniform manner compared to the 1st method. Cells are allowed to settle at the bottom of a syringe, and are then delivered into the hollow lumen at constant flow controlled by a syringe pump. (C) Immunostaining of TY10 monolayers: endothelial cell marker PECAM-1/CD31, transporters Glut-1 and transferrin receptor (TfR), and tight junction proteins ZO-1, Occludin and Claudin-5. Staining for laminin provides evidence that TY10 cells deposit extracellular matrix while in culture. Scale bar, 100 mum. (D) Representative image of a chemically fixed sample of TY10 cells after they were grown in the brain microvessel-on-a-chip with medium flowing from left to right at 1 mµL/min for 7 days. Volumetric image was obtained using a spinning disk confocal microscope. Maximum z-projection is shown for a sample stained with DAPI (nuclei, blue) and phalloidin (actin, green). Scale bar, 100 mum. (D') Representative image of a chemically

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Experimental details: FIGURE 5 Overlayer images of confocal stacks of Caco-2 BBe cell layers showing green-fluorescent cytoskeleton (A-D) and blue-fluorescent nuclei (E-H) , fluorescence images of red-fluorescent ZO-1 tight-junction proteins (I-L) and apoptosis of green-fluorescent membrane-damaged cells with red-fluorescent nuclei (M-P) . For comparison, a fluorescent image of apoptotic cells is shown in Supplementary Figure 3 . (A,E,I,M) 24 h Caco-2 BBe cell layers grown in the absence of an E. coli challenge. (B,F,J,N) Caco-2 BBe cell layers in the presence of an E. coli (10 4 mL - 1 ) challenge before the occurrence of the TEER maximum, i.e., at 4 h. (C,G,K,O) Caco-2 BBe cell layers grown in the presence of an E. coli (10 4 mL - 1 ) challenge at the TEER maximum, i.e., at 8 h. (D,H,L,P) Caco-2 BBe cell layers grown in the presence of an E. coli (10 4 mL - 1 ) challenge after the occurrence of the TEER maximum, i.e., after 24 h.

Supportive validation

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Experimental details: Figure 5 The intestinal barrier is maintained by AC in ob/ob mice. ( A ) Relative mRNA expression of tight junction proteins and CD36 between ob+/+ -Ctrl and ob/ob -Ctrl mice; ( B ) The intestine was examined using hematoxylin and eosin staining and immunohistochemical staining of CD36. Magnification, 40x. Scale bars are 20 mum. The negative control for immunohistochemistry has been marked NC; ( C ) The intestine was examined using immunohistochemical staining for ZO-1. Magnification, 40x and 100x; scale bars are 50 mum and 10 mum, respectively. All data are expressed as mean +- SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Non-significant; ns. n = 4-8 mice in each group.

Supportive validation

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Experimental details: Figure 6 EEAC decreases intestinal permeability in the Caco-2 cell barrier model. ( A ) Cell viability assay of Caco-2 cells treated with 0-3000 muL/mL EEAC. ( B ) Effect of EEAC on intestinal permeability was measured using the trans-epithelial electrical resistance (TEER) assay. ( C ) Relative mRNA expression of tight junction proteins and PPARgamma were compared using controls (Ctrl). ( D ) Immunofluorescence staining of ZO-1 (green) of Caco-2 cells treated with or without EEAC. Nuclei were stained with DAPI (blue). The negative control is marked as NC. Scale bars, 20 mum. All data are expressed as mean +- SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Non-significant; ns. All experiments were repeated thrice ( n = 3).

Supportive validation

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Experimental details: Figure 3 Characterisation of olfactory and respiratory primary cells in comparison to the standard cell line RPMI 2650. Monolayers are necessary to evaluate transport over the epithelial layer in the nasal mucosa: 14 um sections were made of olfactory epithelium primary cells (OEPC, A ), respiratory epithelial primary cells (REPC, B ) and RPMI 2650 ( C ) grown on a cell insert membrane for 21 days. Morphological features such as tight junctions and the formation of cilia are important influencing factors in investigations of drug permeation and clearance studies. Acetylated alpha-tubulin is a common marker for cilia [ 54 ]. IF double-staining of acetylated alpha-tubulin and the tight junction marker zonula occludens-1 (ZO-1) of ALI cultures of OEPC ( D ), REPC ( E ) and RPMI 2650 ( F ) after 21 days of incubation were made. An additional feature of mucosal cells is the ability to produce mucus. The marker protein mucin 5AC was used in this work, because the olfactory mucosa is to be simulated above all to investigate nose-to-brain transport. Again, IF was performed in OEPC ( G ), REPC ( H ) and RPMI 2650 ( I ). Scale bars: 100 um.

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Experimental details: Fig 2 Representative immunohistochemistry images for Claudin 2 ( A-C ), Claudin 4 ( D-F ), and ZO-1 ( G-I ). Pigs were either PRRSV naive and fed ad libitum (Ad: A, D, and G ), PRRSV naive and pair-fed to PRRS+ pigs intake (PF: B, E, and H ), or challenged with PRRSV (PRRS+: C, F, and I ). Pigs were euthanized at days post inoculation (dpi) 17. Staining was limited to the epithelial cells, with higher stain density at cell membranes.

Supportive validation

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Experimental details: Fig. 2 Granulosa cell intercellular junctions are less abundant in Rspo2 Tg/Tg follicles. a Immunodetection analyses of FOXL2 and AMH (granulosa cells) at 21d in wild-type (WT) and Rspo2 Tg/Tg transplanted ovaries. In Rspo2 Tg/Tg ovaries, FOXL2 is still expressed in granulosa cells, but AMH expression is impaired compared to wild-type. b Immunodetection of LAMA1 (basal lamina), CDH2 (adherens junctions), and TJP1 (tight junctions) at 8d in WT and Rspo2 Tg/Tg transplanted ovaries. Follicles are surrounded by a continuous basal lamina in both genotypes as evidenced by LAMA1 immunostaining. CDH2 and TJP1 signals are decreased, notably at the interface between the oocyte and granulosa cells, indicating a deficit of cohesion in Rspo2 Tg/Tg follicles. Scale bar, 20 um. c This result is corroborated by quantitative analysis of CDH2 and TJP1 signal intensity on confocal sections. Data are presented as mean +- SEM. n = 5 WT and n = 9 Rspo2 Tg/Tg follicles from one transplanted ovary of each genotype. Student's t test, unpaired two sided (* p < 0.05; ** p < 0.01; *** p < 0.001).

Supportive validation

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Experimental details: Figure 3 BZA increases angiogenesis and decreases BSCB destruction in vitro. ( A ) Representative immunofluorescent images (scale bar = 20 mum) of tight junction proteins such as ZO-1 and occludin and angiogenesis markers including ANGPT-1, vWF, and alpha-SMA examined in hCMEC/D3 cells. Quantitative fluorescence intensity for ( B ) ZO-1, ( C ) occludin, ( D ) ANGPT-1, ( E ) vWF, and ( F ) alpha-SMA. Data represent mean +- SEM ( n = 3 ( A , C ), n = 4 ( D ), n = 6 ( F ); performed in triplicate). ** p < 0.01, *** p < 0.001 (LPS-treated vs. control, DMSO), ## p < 0.01 (LPS-treated vs. LPS + BZA 10 muM), $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 (LPS-treated vs. LPS + BZA 20 muM) , NS = Not significant, one-way ANOVA followed by the Bonferroni test.

Supportive validation

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Experimental details: Figure 7 Ruscogenin attenuated the tight junction injury via regulating the expression of TRAP1. The expression of TRAP1, occludin, claudin-5, ZO-1, ki67, and E-cadherin expression in the liver cells stimulated by colorectal liver metastasis via regulating the expression of TRAP1. Representative images on TRAP1 (green), occludin (green), claudin-5 (green), ZO-1 (green), E-cadherin (red), and ki67 (green) in the liver tissues stimulated by colorectal liver metastasis were captured by immunofluorescence staining. Bar =50 um. TRAP1, tumor necrosis factor receptor associated protein 1; ZO-1, zonula occludens 1.

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Experimental details: The confocal imaging of bEnd.3 cells. Nuclei staining (blue). ZO1 staining (yellow). F-actin staining (red). Scale bar = 10 um. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Supportive validation

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Experimental details: Figure 1 P5 Nfix -/- mice demonstrate abnormal localisation of key adhesion proteins to the cell junction. ( A , B , M , N ) Images of brain lateral ventricles from P5 Nfix +/+ ( A ) and Nfix -/- ( B ) mice marked with antibodies against ependymal marker FOXJ1 ( A , B ) or Vimentin ( M , N ) (purple), tight junction protein aPKCzeta ( A , B ) or ZO-1 ( M , N ) (green) and nuclear marker DAPI (grey) displayed separately and merged for each genotype. ( C , O ) Average fluorescence intensity of aPKCzeta ( C ) and ZO-1 ( O ) across the cell junctions of Nfix -/- (red) and Nfix +/+ (blue) ependymal cells. The peak fluorescence intensities of aPKCzeta and ZO-1 are lowered in Nfix -/- mice compared to controls. ( D , P ) Maximum fluorescent intensities of aPKCzeta ( D ) and ZO-1 ( P ) across the cell junctions of Nfix -/- (red) and Nfix +/+ (blue) ependymal cells. Maximum fluorescent intensity of aPKCzeta is significantly reduced in Nfix -/- mice compared to controls. ( E , F , I , J ) Images of brain lateral ventricles from P5 Nfix +/+ ( E , I ) and Nfix -/- ( F , J ) mice marked with antibodies against ependymal marker vimentin (purple), adherens junction protein beta-Catenin ( E , F ) or P120-Catenin ( I , J ) (green) and nuclear marker DAPI (grey) displayed separately and merged for each genotype. ( G , K ) Average fluorescent intensities of beta-Catenin ( G ) and P120-Catenin ( K ) across the cell junctions of Nfix -/- (red) and Nfix +/+ (blue) ependymal cells. The peak fluores

Supportive validation

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Experimental details: Figure 5 MCF7 cells treated with NFIX knockdown lentivirus demonstrate reduced localisation of adhesion proteins. ( A , B ) Images depicting MCF7 cells treated with scramble RNA lentivirus (scramble, A ) or NFIX shRNA lentivirus (NFIX KD, B ) and stained with antibodies against tight junction marker aPKCzeta (green), adherens junction marker beta-Catenin (purple) and nuclear marker DAPI (grey) displayed as separate panels or merged. ( C , D ) Images depicting NFIX KD ( C ) and scramble ( D ) cells stained with antibodies against adherens junction marker P120-Catenin (green), tight junction marker PAR3 (purple) and nuclear marker DAPI (grey) displayed as separate panels or merged ( E , F ) Images depicting NFIX KD ( E ) and scramble ( F ) cells stained with antibodies against adherens junction marker E-Cadherin (green), tight junction marker ZO-1 (purple) and nuclear marker DAPI (grey) displayed as separate panels or merged. ( G , I , K , M , O , Q ) Average fluorescent intensity across the cell junction was plotted for line scanning measurements of aPKCzeta ( G ), beta-Catenin ( I ), P120-Catenin ( K ), PAR3 ( M ), E-Cadherin ( O ) and ZO-1 ( Q ) for NFIX KD cells and controls. Tight junction proteins aPKCzeta and ZO-1 demonstrated reduced fluorescent intensity peaks in NFIX KD cells compared to controls. Tight junction protein PAR3 demonstrated no changes between treatment conditions. Adherens junction proteins beta-Catenin, P120-Catenin and E-Cadherin demonstrated reduced p

Supportive validation

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Experimental details: Tamoxifen treated Nfix iFOXJ1-GFP ependymal cells demonstrate reduced expression of adhesion proteins. ( A , B ) Lateral ventricle sections from Nfix iFOXJ1-GFP mice treated with corn oil ( A ) and tamoxifen ( B ) at 7 dpi labelled with aPKCzeta (purple), ependymal cell marker FOXJ1 (green) and nuclear marker DAPI (grey), displayed separately and merged. ( C ) Average aPKCzeta fluorescent intensity across the cell junction is displayed for tamoxifen treated Nfix iFOXJ1-GFP ependymal cells and controls. Average aPKCzeta fluorescent intensity across the cell junction was increased at the peak in tamoxifen treated Nfix iFOXJ1-GFP ependymal cells compared to controls. ( D ) Maximum aPKCzeta fluorescent intensity is displayed for tamoxifen treated Nfix iFOXJ1-GFP ependymal cells and controls. Maximum aPKCzeta fluorescent intensity is significantly increased in tamoxifen treated Nfix iFOXJ1-GFP ependymal cells compared to controls. ( E , F ) Lateral ventricle sections from Nfix iFOXJ1-GFP mice treated with corn oil ( E ) and tamoxifen ( F ) at 7 dpi labelled with beta-Catenin (purple), ependymal cell marker Vimentin (green) and nuclear marker DAPI (white), displayed separately and merged. ( G ) Average beta-Catenin fluorescent intensity across the cell junction is displayed for tamoxifen treated Nfix iFOXJ1-GFP ependymal cells and controls. Peak beta-Catenin fluorescent intensity is reduced in tamoxifen treated Nfix iFOXJ1-GFP mice compared to controls. ( H ) Maximum beta-Catenin f

Supportive validation

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Experimental details: FIGURE 5 Histological changes of hematoxylin-eosin staining assay in colonic and cecum tissue in HFD mice induced by P. vulgaris extract (500 mg/kg, PHAS 500) treatment. Micrographs are representative pictures with an original magnification of x20 (n = 4). (A) Colon tissue from the control group. (B) Colon tissue of HFD mice, showing severe goblet cell hyperplasia and mild epithelial hyperplasia with low-grade dysplasia. (C) Colon tissue of HFD + PHAS group, showing moderate goblet cell hyperplasia. (D) Cecum tissue from control mice. (E) Cecum tissue of HFD mice, showing severe goblet cell hyperplasia and mild epithelial hyperplasia with low-grade dysplasia. (F) Cecum tissue of HFD + PHAS group, showing moderate goblet cell hyperplasia. Protective effect of P. vulgaris extract (500 mg/kg, PHAS 500) treatment on colonic and cecum barrier integrity in HFD mice: in particular, immunoblot representatives of occludin expression in the colon (G) and cecum (I) and ZO-1 in the colon (H) and cecum (L) were showed. The levels are expressed as the density ratio of target to beta-actin. Data are expressed as means +- SEM ( n = 6). ** p < 0.01 vs. STD; # p < 0.05 and ## p < 0.01 vs. HFD.

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Experimental details: Figure 2 CCL7 knockdown suppresses EMT and mobility of LUAD cells. mRNA (a) and protein (b) levels of CCL7 in H1299, A549, H358 (low-invasive), and H125 (high-invasive) cells detected by qPCR and WB analyses; mRNA (c) and protein (d) levels of CCL7 in A549 and H125 cells after overexpression plasmid or shRNA transfection determined by qPCR and WB analyses; mRNA (e) and protein (f) levels of epithelial marker ZO-1 and mesenchymal markers Twist1, Snai1, and Slug in A549 and H125 cells examined by qPCR and WB analyses; (g) 24-h migration rate of A549 and H125 cells analyzed by the scratch test; (h) invasiveness of A549 and H125 cells analyzed by Transwell assay. Repetition = 3. ** p < 0.01.

Supportive validation

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Experimental details: Fig. 1. CHD4 is required during the fourth day of development. (A) Representative composite spinning-disc images of anti-CHD4 (magenta) and SIN3A (green; used as a control) staining in wild-type and Chd4 -/- 2-, 4-, 8- and 16-cell embryos. Images are representative of eight 2-cell embryos, 12 4-cell embryos, 11 8-cell embryos (including both Chd4 -/- and Chd4 Delta / Delta embryos) and four 16-cell embryos. Scale bars: 33 um. (B) Composite confocal images of DAPI (blue) and CHD4 (magenta) staining in wild-type (WT) and Chd4 -/- (KO) 3.5 dpc embryos. Scale bars: 50 um. KO images are representative of >20 mutant blastocysts (including both Chd4 -/- and Chd4 Delta / Delta ). (C) Phase contrast images of wild-type (WT) and Chd4 -/- (KO) embryos flushed at different times on the fourth day post coitus (dpc). Scale bars: 50 um. KO images are representative of 16 Chd4 -/- embryos. (D) Average number of contractions per embryo genotype observed during live imaging (see supplementary material Movies 1-3 ). Each circle indicates the number of contractions for a single embryo of the indicated genotype. (E) Representative confocal 3D projection images of early 3.5 dpc embryos of the indicated genotypes stained for the indicated markers. KO images are representative of six embryos. Scale bars: 50 um. (F) Two representative images per genotype of late 3.5 dpc embryos stained for E-cadherin (white) and DAPI (blue). Each image is a composite of four to six stacks, wh

Supportive validation

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Experimental details: FIGURE 2 Influence of WY-14643 and PPARalpha antagonist GW6471 on effects of OC6 treatment on the protein expression of tight junction proteins ZO-1, occludin and claudin-5 of cerebEND cells. OC6 treatment of cerebENDs accords to 4 h OGD treatment with medium supernatants derived from 4 h OGD-treated C6 cells, whereas NC6 treatment means cerebEND cells incubated for 4 h under normoxic conditions with medium supernatants of 4 h normoxic treated C6 cells. Results for ZO-1 (A) , occludin (B) and claudin-5 (C) were displayed. Densitometric analyzed protein expression of total cell lysates were compared to RIPA and triton-X 100 fractions. Moreover, protein distributions [%] between RIPA and triton-X 100 fractions were presented. Values were related to beta-actin and according representative western blot images are depicted. Dotted lines indicated cuts of images of the same blot due to presentation reasons of selected bands. Statistical significance was labeled with * vs. NC6 and # vs. OC6 ( p < 0.05). Data are presented as means +- SEM ( n = 3-8).

Supportive validation

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Experimental details: FIG 4 GSNO contributes to the maintenance of epithelial tightness of rotavirus-infected polarized Caco-2 cells and induces increased expression of ZO-1. (A) Polarized Caco-2 cells (>450 Omega/cm 2 ) were stimulated with GSNO for 24 h and infected with rotavirus. At 6 h p.i., the apical medium was replaced with medium containing 4-kDa FITC-dextran (2 mg/Transwell insert), and samples were obtained from the basolateral side at 22 h p.i. to measure passage of FITC-dextran. The controls were noninfected cells with and without GSNO and infected cells without GSNO. Basolateral samples were analyzed for FITC-dextran by spectrophotometry (494/518 nm), and values are presented as the optical density (OD) values and means with SD. * , P < 0.05, unpaired t test ( n = 5 or 6). (B) GSNO increases ZO-1 tight junction protein in Caco-2 cell monolayers. Caco-2 cell monolayers cultured on Lab-Tek II chamber slides to near confluence with and without 24-h exposure to GSNO were stained for ZO-1 expression (red; Alexa Fluor 594). Confocal images were captured and the mean intensities on single-cell circumference were measured using ImageJ. (C and D) Monolayers of GSNO-treated (C) and untreated (D) Caco-2 cells. Data are mean fluorescence intensities and means with SD. ***, P < 0.001, Mann-Whitney test ( n = 17). (E and F) Deconvolved images visualized in ortho-mode with XZ and YZ intensities of GSNO-treated (E) and untreated (F) Caco-2 cells. The XZ and YZ selections in each image are positioned

Supportive validation

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Experimental details: Fig. 2 Non-immunohistochemical staining with tissue clearing on formalin-fixed brain tissues. a Colour depth-coded image with maximum intensity projection of a piece of 1-mm-thick medulla block stained with cresyl violet and cleared with OPTIClear (z-stack depth = 228.83 um). Lower image showing a zoomed and tilted view of area outlined by the white box. b A piece of frontal cortical block immunostained for ZO-1 (green) and counterstained with DyLight 649-labelled Lycopersicon esculentum lectin (red). c DiI crystals were inserted into a cerebellar folium to trace the fibres up to the granular layer, followed by tissue clearing with OPTIClear for 6 h at 37 degC. Most of the fibres traced were mossy fibres. The inset shows the gross appearance of the sample with red crystals of DiI inserted within the OPTICleared sample. d Enlarged view of the white boxed region in a. with colour depth-coding (Z-depth 151.09 mum). e Z -stack images of dendritic spine-like projections visualised in areas outlined by white boxes. Scale bars, 1 mum

Supportive validation

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Experimental details: Figure 4 Effects of SA on small-intestinal injury in mice with MCD diet-induced steatohepatitis. ( A ) Histology was carried out using H&E staining (magnification, 100x and 200x), and representative images are shown. The height of five intact and well-oriented villi per sample was measured using ImageJ and the average value used as data. Results are the mean +- SEM for 4-6 mice per group. + p < 0.01. ( B ) Mucus production in the small intestine was detected using PAS staining (magnification, 100x and 200x), and representative images are shown. ( C ) Small-intestine sections were stained with ZO-1 (green) and DAPI (blue). Representative immunofluorescence images are shown. Magnified images represent high-magnification images of the boxed area.

Supportive validation

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Experimental details: Figure 3 Effect of miR-143 on the permeability of endothelial cells in vitro. (a) Methamphetamine increased the permeability of the HBMECs. (b) Effect of methamphetamine on the expression of claudin-5, occludin, and ZO-1 in HBMECs evaluated using western blot analysis. (c,d) Effect of miR-143 on the expression of tight junction proteins (c) and the permeability of HBMECs (d) . (e,f) Effect of anti-miR-143 on the expression of tight junction proteins (e) and the permeability of HBMECs (f) . All data are presented as the mean +- SD of three individual experiments. **p < 0.01 vs. the control group using Student's t-test. (g) Anti-miR-143 attenuated the methamphetamine-induced increase in the permeability of HBMECs. *p < 0.05 and **p < 0.01 vs. the anti-miR-con/control; ### p < 0.001 vs. the anti-miR-con/meth group using one-way ANOVA followed by the Holm-Sidak test. Meth, methamphetamine.