34-1700

antibody from Invitrogen Antibodies

Targeting: CLDN3 C7orf1, CPE-R2, CPETR2, HRVP1, RVP1

Provider product page for 34-1700

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Antibody data

Antibody Data
Antigen structure
References [190]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [2]
Other assay [93]

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Product number: 34-1700 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 3 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody is specific for the 22 kDa Claudin-3 protein. Reactivity has been confirmed with mouse liver and lung homogenates, canine MDCK and human MCF-7 cell lysates by western blotting, and with formalin-fixed, paraffin-embedded (FFPE) human normal colon and colon cancer tissues by immunohistochemistry. For best results in immunohistochemistry with formalin-fixed, paraffin-embedded (FFPE) tissues, heat induced epitope retrieval (HIER) with citrate buffer, pH 6.0, is required prior to staining.
Reactivity: Human, Mouse, Canine
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

CLDN3 protein structure - 34-1700 shown in red.

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Experimental details: Fig 5 Localization of claudins in z-axis plane in control and claudin-2 knockout cells. Immunofluorescence analysis of claudins and occludin in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1) in z-axis plane. The signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells. Scale bar = 5 mum.

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Experimental details: Fig 4 Effects of claudin-2 knockout on the localization of other claudins. (A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 mum. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods , and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.

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Experimental details: Fig 5 Localization of claudins in z-axis plane in control and claudin-2 knockout cells. Immunofluorescence analysis of claudins and occludin in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1) in z-axis plane. The signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells. Scale bar = 5 mum.

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Experimental details: Fig 8 Effects of claudin-2 re-expression on the localization of other claudins in claudin-2 knockout cells. (A) Immunofluorescence analysis of claudins and FLAG in co-culture of claudin-2 knockout cells and F2 or F4 clones. Claudin-1, -3, -4, and -7 staining at TJs in F2 and F4 clones was weaker than those in claudin-2 knockout cells. Scale bar = 10 mum. (B) Quantification analysis of the signal intensity of claudins at TJs in F2 and F4 clones. The signal intensity of claudin-1, -3, -4, and -7 at TJs in F2 and F4 clones was compared with that in claudin-2 knockout cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with claudin-2 knockout cells.

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Experimental details: Fig 11 Localization of claudins at TJs between control and their respective knockout cells. (A-E) Immunofluorescence analysis of claudins and ZO-1 or ZO-3 in MDCK II cells transfected with the TALEN constructs for claudin-1, -2, -3, -4, or-7 gene knockouts. After transfection, cells were subcultured on filter inserts for 4 days before the analysis of claudin-2 and -3 (B and C), and for 2 days before the analysis of claudin-1, -4, and -7 (A, D, and E). (F) Immunofluorescence analysis of claudin-2 and ZO-1. TALEN constructs for the claudin-2 gene knockout were transfected into MDCK II cells, which were subcultured on filter inserts for 2 days before analysis. (G) Immunofluorescence analysis of claudin-2 and ZO-1 in a co-culture of wild-type and claudin-2 expressing MDCK I cells. Cells were cultured on filter inserts for 4 days before analysis. Scale bar = 10 mum.

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Experimental details: Figure 4 Western blot and immunofluorescence analysis of parental bipotent cells and MPCs using various markers from the signature of claudin-low breast cancer subtype. ( A ) Western blot analysis of cell lysates from exponentially proliferating bipotent cells and MPCs were analyzed for expression of indicated markers using specific antibodies. Breast cancer cell lines MDA-MB-231, MCF-7, and Hs578T were used as controls. ( B ) Immunofluorescence staining using CD24 antibody (red), nuclei (blue) represent DAPI staining.

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Experimental details: FIGURE 6: Rab25 depletion alters the transepithelial resistance and claudin expression. (A) TER was measured in all cell lines from day 3 to day 15 on Transwell filters. During this period, the media were changed every day, and TER was measured in 24-well Transwell inserts. Results are expressed in ohm-cm 2 and are representative of three separate experiments. (B) Equal amounts of total protein lysates (20 mug) from Control, Rab25KD, and Rescue cells were analyzed by Western blots for tight junction proteins. The blots were subsequently stripped and reprobed for beta-actin as a loading control. The results are representative of three separate experiments.

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Experimental details: Figure 6 Expression of TJ proteins in transmigrating cells favors their passage across RBMECs. A) Analysis by Western blots of the expression of occludin, claudins 1 to 5 and CRTAM in NIH-3T3and L-fibroblasts, ReNcells CX and RBMECs. B) The transmigration of NIH-3T3 and L-fibroblasts acrossRBMECs is independent of the CM present in the basal compartment, but is significantly higher inNIH-3T3 fibroblasts that express claudin-2 than in L-fibroblast that do not express TJ proteins.N = 3, F(1,6) = 232.22, ***P

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Experimental details: Figure 4. Expression and localization of tight junction (TJ) proteins in cell lines expressing typical hepatocyte polarity. Fully polarized cultures were examined. ( A ) western blot. Each lane was loaded with a cell lysate aliquot containing 25 mug total protein. ( B ) RT-PCR. For western blot and RT-PCR, experiments were performed three times with lysates or RNA preparations from two to three independent cultures; one representative experiment is shown. ( C ) Summary of the localization of ten TJ proteins. Experiments were performed at least three times on three independent cultures. ( D ) Images of the localization of some TJ proteins; for each case, the corresponding phase-contrast image is given on the left. Some bile canaliculi (BC) are indicated by arrows (short arrows for BC formed between two cells, long arrows for extended BC). Bar, 10 mum

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Experimental details: Figure 3 Protein expression (Western blot) of claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-8 after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC). (a) Protein expression relative to respective controls [means +- SEM]. Samples were taken after 8 h. No differences were observed between treatment groups. N = 3 independent experiments per bar. (b) Exemplarily, data of one Western blot is shown. 1 = control (8 h), 2 = Ecf (8 h), 3 = ETEC (8 h), 4 = Ecf (8 h) + ETEC (6 h), and 5 = Ecf (10 h) + ETEC (8 h). Sample 4 was included as a control to rule out effects of longer total bacterial incubation (preincubation) and was not included in the statistical analysis shown in (a).

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Experimental details: Figure 10. Impact of silencing claudin-3 on Can 10 polarity. Cells transfected either by Lipofectamine with no siRNA, scrambled siRNA or three different claudin-3 siRNA were examined 48 h after tranfection. ( A ) RT-qPCR of one representative experiment. Claudin-3 mRNA level is expressed as the percentage of the level of cells treated with no siRNA. ( B ) western blot. An immunoblot corresponding to the experiment shown in A is presented on the left. Densitometric analysis of immunoblots obtained from three independent experiments is on the right. Claudin-3 level, normalized with alpha-tubulin as internal control, was expressed as the percentage of claudin-3 level of cells treated with no siRNA. ( C ) Analysis of cells forming bile canaliculi. Values are means of three independent experiments. ( D ) Co-localization of claudin-3 with ZO-1, and claudin-5 with occludin, studied by confocal microscopy. In each case, the localization of each tight junction protein (compilation of 20-27 xy sections) is shown, followed by a composite image (interferentiel contrast image with the merge image) (right). For cells treated with claudin-3 siRNA2, xz sections taken as indicated (thin white lines) on the corresponding xy compilations are also presented. Bar, 10 mum.

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Experimental details: Figure 2 Time course of urinary claudin-3:creatinine ratio of two representative of the last five children undergoing spinal fusion surgery.

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Experimental details: Fig. 3 Western blot analysis and cellular distribution of claudin-3 in choroid plexus and cerebral parenchyma during rat brain development. a Representative Western blot of LVCP and 4VCP dissected from E19, P2, and adult animals (10 mug per lane). Cld-3 protein levels decreased in both types of CP during development ( upper panel band at 22 kDa). Actin used as a loading indicator is shown in the lower panel (42 kDa band). b - g Cld-3 immunoreactivity ( red ) in the developing and adult brain. Double immunolabeling with anti-RECA-1 Ab ( green ) allowed visualizing choroidal and parenchymal vessels (e.g. arrowheads in b , g ), and DAPI was used for nuclei staining. During perinatal stages of development, immunodetection revealed a distinct signal associated with the CP epithelial cell membranes as illustrated for LVCP in E19 ( b ), or in P2 animals ( c ). The inserts in b and c highlight the typical honeycomb pattern of choroidal epithelial cell junctions. The area delimited by the rectangle in c , is enlarged in d , which shows Cld-3 in a parenchymal vessel of the developing brain ( i Cld-3 ( red ), ii RECA-1 ( green ) and iii merge staining). Cld-3 immunoreactivity was observed at the epithelial junctions in CPs of P9 animals ( e ), and could still be detected in adult CP ( g ). f A negative control (NC), run in the absence of primary antibody, for a P9 animal ( arrow CP). Scale bars 20 mum. Abbreviations as in Fig. 2

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Experimental details: Figure 5 GC-C is essential to maintain intestinal barrier function during C. rodentium infection. A : GC-C +/+ and GC-C -/- mice were given a FITC-dextran tracer by oral gavage on day 4 p.i. and serum fluorescence was measured four hours later. Relative to naive mice as well as infected GC-C +/+ animals, GC-C -/- mice have a substantial and significant increase in tracer movement from the gut lumen into the blood indicating a loss of intestinal barrier function. (* P

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Experimental details: Figure 7. Settlement of tight junction (TJ) proteins and paracellular permeability throughout the polarization of cell lines expressing typical hepatocyte polarity. ( A ) Settlement of constitutive (left) and associated- (right) TJ proteins at TJs sealing bile canaliculi (BC), analyzed by immunolocalization. ( B ) Paracellular permeability throughout the settlement process. Values are means of three independent cultures of each cell line ; 11-3 values (not shown) were similar to those shown here for WIF-B9 cells. In Can3-1 cells, claudin-4 was not settled at TJs as the other TJ proteins, and was present at the whole plasma from the first day. ( C ) Localization of claudin-3 with ZO-1 on day 3, and of claudin-2 with ZO-1 on day 7, by confocal microscopy in Can 10 cells. The localization of the TJ proteins (compilation of 21-24 xy sections) is shown, followed by the merge image and the composite image (interferentiel contrast image + merge image), Note that on day 3 claudin-3 and ZO-1 were completely settled at the TJs sealing bile canaliculi, whether bile canaliculi were formed between two cells or more. By contrast, claudin-2 was not completely settled on day 7. It was either absent or partially present at the TJs where ZO-1 localized. Bar 10 mum,

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Experimental details: Figure 2 Overexpression of FoxN1 mediated by uCreER T+ in juvenile mice induced medullary-biased abnormality of thymic microstructure. Representative immunofluorescence staining shows the thymuses of FoxN1 Tg uCreER T+ juvenile (~20 days after birth) mice (right panels) had infused thymic medullary islets (top panels), an increase in UEA-1 + and Cld3,4 + TECs (panels in middle two rows), an increase in Aire + mTECs (bottom panels), and dimness of 5 beta t + bright dendritic-shaped spots (second row from top), compared to their normal littermate controls (left panels). This experiment was repeated three times with at least three animals in each group producing consistent results

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Experimental details: Fig. 1 Knockdown of claudin-7 in HCC827 lung cancer cells. a Representative phase and green fluorescence images of live control and claudin-7 KD cells. Both control and claudin-7 shRNA lentivirus constructs contain a GFP expression sequence. b Top panel is the anti-claudin-7 immunofluorescence staining of control and claudin-7 KD cells. Claudin-7 level was dramatically decreased in claudin-7 KD cells. The bottom panel is the anti-GFP immunofluorescence staining. The cells were fixed with 100 % methanol, which lead to GFP proteins leaking out of the cells so that the green fluorescence shown in the top panel was only the claudin-7 signal. c Western blot shows the diminished level of claudin-7 in the KD cells ( left ). The statistical analysis of claudin-7 expression in control and KD cells from five independent experiments is shown on right. * P < 0.05. d Protein expression levels of E-cadherin, claudin-1, -3, and -4 in control and KD cells. Only claudin-4 expression level was increased in KD cells. GAPDH served as a loading control

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Experimental details: Figure 1 Dental phenotype of Claudin-3 KO mice. (A) Claudin-3 mRNA expression was analyzed in mouse tissues by RT-PCR using forward primer 5'-ATG TGG CGC GTT TCG-3' and reverse primer 5'-GCG AGT CGT ACA TTT TGC-3' for Claudin-3 (124 bp), and forward primer 5'-TGT GTC CGT CGT GGA TCT GA-3' and reverse primer 5'-TTG CTG TTG AAG TCG CAG GAG-3' for Gapdh (150 bp). A representative gel image of the selected tissues showed Claudin-3 expression in mouse in post-natal day 3 tooth germ, liver and heart. Identification of claudin-3 in the continuously growing incisor was performed by immunofluorescence using claudin-3 antibody (#34-1700, Invitrogen) at 1/100 dilution and a Goat anti-Rabbit antibody (#R-6394, Invitrogen). Claudin-3 was localized at the distal end of secretory ameloblasts (am). Protein expression was also observed at the basal end of the cells ( n = 3 per group). (p) pulp. (B) Recapitulative schema of claudin TJ proteins expression at the apical end of secretory ameloblasts. Claudin-3, -16, and -19 were shown to be expressed at TJ level of ameloblast secretory ends during enamel formation in the mouse. (C) 3D volume rendering from Micro-CT data showed severe enamel loss on lingual molar cusps in Claudin-3 KO mice. Quantitative analysis confirmed a significant lower enamel volume in Claudin-3 KO mice when compared to WT (0.022 vs. 0.120 mm 3 respectively) ( n = 8 per group). In WT mice, incisor mineralization is observed under the t

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Experimental details: Figure 12 Effects of ZO-1 knockout on the localization and expression levels of claudins. (A) Effects of ZO-1 knockout on the localization of claudins. Control and ZO-1 knockout cells of clone 1 were co-cultured on filter inserts. Signal intensity of claudins on lines shown in confocal microscopic images ( arrows ) were analyzed. Claudin-2 fluorescent signal at TJs was increased but claudin-1 and -7 signals at TJs were reduced in ZO-1 knockout cells. Scale bar, 10 um. (B) Immunoblots of claudins in control MDCK II cells and ZO-1 knockout clones. Similar expression levels of claudins were observed in control and knockout cells.

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Experimental details: Fig 3 Effects of claudin-2 knockout on the localization of other TJ proteins and cytoskeleton. (A) Immunofluorescence analysis of claudin-1, -3, -4, -7, ZO-1, occludin, F-actin, and myosin heavy chain II-B (MHC-B) in control and claudin-2 knockout cells. Claudin-1 and -7 showed a tendency to be more clearly localized at TJs in claudin-2 knockout cells. Scale bar = 10 mum. (B) Immunoblots of claudin-1, -3, -4, and -7 in control MDCK II cells and claudin-2 knockout clones. Similar expression levels of claudin-1, -3, -4, and -7 were observed in control cells and knockout clones.

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Experimental details: Fig. 8 Immunohistochemical detection of claudins in choroid plexuses of developing human brain. Immunoreactivity of Cld-1, -2, and -3 is presented on the left, central, and right panels , respectively, with hematoxylin counterstaining. a Staining of a WD8 fetal brain. Cld-1 labeling, observed in both LVCP and 4VCP, was continuous and localized apically at intercellular junctions of the epithelium ( arrows in inserted high magnification micrograph ). Cld-2 immunoreactivity displayed a similar apical, but patchy pattern in both LVCP and 4VCP. The higher magnification ( insert in LVCP) illustrates the discontinuous labeling ( arrows ). Cld-3 was also detected in the epithelium of both CP ( arrows ). Inserts are higher magnifications highlighting the continuous staining pattern. b Staining of a WD22 fetal brain. Cld-1 immunoreactivity was present at all epithelial junctions (shown in 4VCP). A continuous apical labeling of the epithelium was observed for Cld-2 (LVCP is shown). Cld-3 labeling was faint but continuous in LVCP epithelial cells. c Staining of a WD39 fetal brain. A distinct and continuous labeling of the choroidal epithelium was observed for all Cld-1 (shown for LVCP), -2 (shown for 4VCP), and -3. The continuous signal for Cld-3 was only associated with the epithelium ( arrows ) and not with stromal vessels ( arrowheads in LVCP). The insert shows a similar Cld-3 staining for 4VCP. Abbreviations as in Fig. 2 . Scale bar 50 mum

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Experimental details: FIGURE 6 Double-immunostains for the tight junction proteins (green) claudin-3 (a) and claudin-19 (b) and for the adaptor protein ZO-1 (red, a,b ). The nerve fiber layer (NFL) shown at high magnification in the right images, is strongly positive for both claudin antibodies. Immunoreactivity for ZO-1 was also detected in the NFL, as well as in the outer limiting membrane (OLM). In the NFL, claudin and ZO-1 stains largely overlap with the claudins showing more continuous label strands often but not always running parallel to nerve fibers. Scale bar 20 mum for low power, 5 mum for high power images.

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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: Figure 1 Epithelial barrier formation in IPEC-J2 cells grown in control medium and in the presence of 10 nM ouabain (Oua). After 7 days of culture, transepithelial resistance (TER) values were determined every 2-3 days using an EVOM volt-ohmmeter; after 19 days, cells were used for subsequent analysis. ( a ) Transepithelial resistance (TER) ( n = 6 for each group). ( b ) The expression level of total cSrc (central plot, the relative expression of cSrc protein shown as a percentage of average level in control samples taken for 100%) and the cSrc-kinase activation by phosphorylation (right plot, shown as a ratio between immunoblot intensity corresponding to phosphorylated pcSrc over total cSrc, as it is shown in the representative immunoblots in left panel) ( n = 5 for each group). ( c ) Western blot analysis of claudin (CLDN) and tricellulin expression ( n = 6 for each group); left panel shows representative immunoblots. Original images for Western blots using Stain-Free gels as a loading control are shown in Supplemental Materials . The number of symbols corresponds to the number of samples. One-way ANOVA with Dunnett correction: * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the corresponding control.

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Experimental details: Figure 3 Chronic exposure to ouabain (Oua) differently alters claudin expression in rat jejunum ( a ) and colon ( b ). Rats were intraperitoneally injected with ouabain (1 µg/kg) for 4 days. Western blot analysis of claudins (CLDN) expression ( n = 4 for each group); left panel shows representative immunoblots. Original images for Western blots using Stain-Free gels as a loading control are shown in Supplemental Materials . The number of symbols corresponds to the number of samples. One-way ANOVA with Dunnett correction: * p < 0.05 and ** p < 0.01 compared with the corresponding control (vehicle treated group).

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Experimental details: Figure 5 Tofacitinib treatment ameliorates intestinal architecture and decreases intestinal epithelial cell proliferation in villin-Cre Hdac1 and Hdac2 deficient mice. Jejunal tissue sections, from wild-type (Ctrl) and villin-Cre Hdac1 -/- ; Hdac2 -/- mice ( Hdac1/2 DeltaIEC) treated without or with Tofacitinib (DMSO or TOFA) for five days, were stained with hematoxylin and eosin (HE) ( A ) (Magnification: 10x) or were stained with an antibody against claudin 3 ( n = 3) (white arrows) ( B ) (Magnification: 20x). ( C ) Wild-type (Ctrl) and villin-Cre Hdac1 -/- ; Hdac2 -/- mice, treated without or with Tofacitinib (DMSO or TOFA) for five days, were injected with BrdU for 2 h. Jejunal tissue sections were stained with an antibody against incorporated BrdU. Magnification: 10x. ( D ) The average number of BrdU-labeled proliferative cells per jejunal crypts was measured ( n = 3, 20-30 crypts each). Results represent the mean +- SEM (* p

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Experimental details: Figure 3 Quercetin attenuated diquat-induced ROS accumulation and reduced protein abundance of tight junction proteins in IPEC-1 cells. IPEC-1 cells were treated as described in Figure 2 . ( A ) DCFH-DA-positive populations were detected by flow cytometry analysis. ( B ) Intracellular ROS levels were determined by fluorescence microscope (magnification x200). ( C ) Intracellular GSH levels were measured. ( D , E ) Protein abundance for ZO-1, ZO-2, ZO-3, occludin, claudin-1, claudin-3, and claudin-4 were determined and analyzed by the Western blot assay. beta-actin was used as the loading control. Representative experimental results were repeated in three independent experiments and values are means +- SEMs, n = 3. Means without a common letter are considered as a significant difference, p < 0.05.

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Experimental details: Figure 4 Effects of whole LP powder, methanol (MetOH) extract, and MetOH extraction residue (MetOH residue) on tight junction protein expression in the colon of dextran sodium sulfate (DSS)-induced colitic mice. Protein expression of zonula occludens (ZO)-1, ZO-2, occludin, claudin-3, and claudin-7 in the colon of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration, as determined by immunoblot analysis. Values are the mean +- SEM ( n = 7). Means without a common letter differ, p < 0.05.

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Experimental details: Figure 5 Effects of whole LP powder, methanol (MetOH extract), and MetOH extraction residue (MetOH residue) on claudin-3 expression in the colon of dextran sodium sulfate (DSS)-induced colitic mice. Immunolocalization of claudin-3 in the colon of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration, as analyzed by immunofluorescence microscopy. Representative images of seven mice in each group are shown.

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Experimental details: Figure 2 The effect of short-chain fatty acids on TNF-alpha/IFN-gamma-induced dysregulation of tight junction proteins. Western blot analyses of ( a ) claudin-1 (CLDN1, 22 kDa), ( b ) claudin-2 (CLDN2, 22 kDa), ( c ) claudin-3 (CLDN3, 22 kDa), ( d ) claudin-4 (CLDN4, 22 kDa), ( e ) occludin (65 kDa), and ( f ) ZO-1 (220 kDa) proteins were performed following stimulation on the basolateral side of the membrane, with the combination of TNF-alpha/IFN-gamma (10 ng/mL), and on the apical side with acetate (ace), propionate (pro), butyrate (but), or succinate (suc) (2 mM). * p < 0.05 compared to medium-treated control; # p < 0.05 compared to TNF-alpha/IFN-gamma group; **** p < 0.0001 compared to medium-treated control.

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Experimental details: Figure 4 The effect of butyrate on IL-13-induced dysregulation of tight junction proteins. Western blot analyses of ( a ) claudin-1 (CLDN1, 22 kDa), ( b ) claudin-2 (CLDN2, 22 kDa), ( c ) claudin-3 (CLDN3, 22 kDa), ( d ) claudin-4 (CLDN4, 22 kDa), ( e ) occludin (65 kDa), and ( f ) ZO-1 (220 kDa) proteins were performed following stimulation on the basolateral side of the membrane with IL-13 (10 ng/mL) and on the apical side with butyrate (but, 2 mM). ** p < 0.01 compared to medium-treated control (Cont); # p < 0.05 compared to IL-13 group.

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Experimental details: 3 FIGURE Immunoblotting using antibodies against zonula occludens (ZO)-1 (A), tricellulin (B), occludin (C), claudin-3 (D), and claudin-4 (E) in intestines undergoing VP only (white bar) and in intestines receiving additional luminal preservation (light gray bar). The black bar denotes the baseline value obtained in the same intestines before cold preservation. Data expressed as mean +- standard error of the mean, * p < .05.

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Experimental details: 4 FIGURE Expression levels of claudin-2 (CLDN2) and claudin-3 (CLDN3) proteins in duodenal mucosa. CLDN2 (22 kDa), CLDN3 (22 kDa), and GAPDH protein expression were evaluated by western blotting in controls ( n = 13) and patients with IBS ( n = 12). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant.

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Experimental details: Fig 6 Localization of claudin-3, E-cadherin, occludin and Na + /K + ATPase under the 'Apical' and 'Basal' conditions in MDCK I cells and barrier function of TJs around the cavities within the multi-layered epithelia. (A and B) Immunofluorescence microscopy for claudin-3 and E-cadherin in z-axis plane under the 'Apical' (A) and 'Basal' (B) conditions in MDCK I cells. Signals of claudin-3 were observed within the multi-layered MDCK I cells under the 'Basal' condition ( arrows ). (C and D) Immunofluorescence microscopy for occludin and Na + /K + ATPase in z-axis plane under the 'Apical' (C) and 'Basal' (D) conditions in MDCK I cells. Signals of occludin were observed within the multi-layered MDCK I cells under the 'Basal' condition ( arrows ). (E and F) A tracer experiment using a primary amine-reactive biotinylation reagent was performed in MDCK I cells cultured under the 'Apical' (E) and 'Basal' (F) conditions. The biotinylation reagent was administered in the basal side for 10 min, and the bound biotin was detected by streptavidin. The biotinylation reagent appeared to stop at TJs around the cavities within the multi-layered epithelia under the 'Basal' condition ( arrows ). Scale bars = 10 mum.

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Experimental details: Fig 9 Effects of apical hyposmolality in claudin-2 knockout MDCK II cells. (A) Time course of P Na and P Cl in claudin-2 knockout MDCK II cell clone (knockout clone 1 in a previous study [ 22 ]). The values of P Na and P Cl were increased and then decreased during 120 min of incubation, and the charge selectivity showed no remarkable change under the 'apical hyposmotic' condition. N = 4 for each experiment. (B) Immunofluorescence microscopy for claudin-3 and ZO-1. The signals of claudin-3 and ZO-1 under the 'apical hyposmotic' condition were similar to those under the 'apical isosmotic' condition. Scale bar = 5 mum. (C) Scanning electron microscopy of claudin-2 knockout MDCK II cells. Scale bar = 2 mum. (D) Time course of P Na and P Cl in claudin-2 knockout MDCK II cells expressing exogenous claudin-2. The clones were established from the claudin-2 knockout clone 1 as described in a previous study (F4 clone in a previous study [ 22 ]). P Na was decreased and P Cl was increased gradually with time under the 'apical hyposmotic' condition. N = 3 for each experiment. (E) Immunofluorescence microscopy for claudin-3 and ZO-1. The protruded signals of claudin-3 and ZO-1 from the lines of cell-cell contacts were observed under the 'apical hyposmotic' condition ( arrowheads ). Scale bar = 5 mum. (F) Scanning electron microscopy of claudin-2 knockout MDCK II cells expressing exogenous claudin-2. Globular structures were observed around cell-cell contacts under the 'apical hyposmotic' c

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Experimental details: FIGURE 2: TJs of Tric -KO, Ocln -KO, and Tric / Ocln -dKO cells appear normal in fluorescence microscopy. (A-D) Ctrl, Tric -KO #1, Ocln -KO #1, or Tric / Ocln -dKO #1 cells were mixed and cocultured with Ctrl-GFP-nls cells (green). Cells were immunostained with rat anti-ZO-1 mAb (A), mouse anti-cingulin mAb (B), mouse anti-claudin-1 mAb (C) or rabbit anti-claudin-3 pAb (D) (red) and stained with DAPI (blue). Scale bars, 20 um. (E) Quantification of fluorescence intensity of claudin-3 at cell-cell junctions. The intensity at each cell-cell junction in the Ctrl (dark blue closed circles), Tric -KO #1 (orange closed circles), Ocln -KO #1 (green closed circles), and Tric / Ocln -dKO #1 cells (magenta closed circles) were normalized to the averaged intensity in the Ctrl-GFP-nls cells (lime-green open circles) and plotted against the length of the cell-cell junctions. n = 158 and 120 (Ctrl), 202 and 134 ( Tric -KO), 160 and 139 ( Ocln -KO), and 209 and 136 ( Tric / Ocln -dKO). *** p < 0.001 in Welch's t test of junction-length-weighted average of the fluorescence intensity.

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Experimental details: Figure 4 Effects of hypoxia and/or heat treatment on TJ/AJ proteins in the co-culture model. Relative protein expression of ZO-1 ( A ), occludin ( B ), claudin-3 ( C ) and E-cadherin ( D ) assessed by Western Blot. All target proteins were normalized to reference protein beta-actin. ZO-1 ( E ), occludin ( F ), claudin-3 ( G ) and E-cadherin ( H ) mRNA levels were assessed by qRT-PCR. The target genes were normalized to housekeeping gene beta-actin. All values were presented as means +- SD (N = 3, n = 3). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni's multiple comparison test. Means without a common letter differ at p < 0.05. OCLD occludin; CLDN3 claudin-3; E-cad E-cadherin.

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Experimental details: 10.1371/journal.pone.0256143.g005 Fig 5 Cellular arrangement (cross sections) of jejunum organoids grown in 2D on Snapwells (r) for 18 days. Immunohistochemical staining (including isotype controls) of Claudin-2 and Claudin-3 was performed. Blue triangles indicate ceklls with positive Claudin-2 signal, orange triangles indicate cells with a positive Claudin-3 signal. Representative figures are shown, chosen from three independent experiments with three technical replicates per staining.

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Experimental details: Figure 3 Confocal laser scanning immunofluorescence microscopy was performed to analyze the localization of ( A ) claudin-1 (green) and ZO-1 (red), or ( B ) claudin-3 (green) and occludin (red) after incubation with TNFalpha for 48 h. Nuclei were stained in blue (DAPI). Claudin-3 shows a strong colocalization with occludin (merge, yellow signal, B) (scale bar: 20 um; n = 4; representative pictures).

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Experimental details: Figure 7 Confocal laser scanning immunofluorescence microscopy of ( A ) claudin-1 (green) and ZO-1 (red), as well as ( B ) claudin-3 (green) and occludin (red), nuclei were stained in blue (DAPI). IPEC-J2 monolayers were treated for 48 h with TNFalpha in the presence or absence of the specific MLCK blocker ML-7. ( B ) The yellow signal in the merged pictures represents the colocalization of claudin-3 and occludin (scale bar: 20 um; n = 4; representative images).

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Experimental details: Figure 8 After IPEC-J2 cells were treated for 48 h with 1000 U/mL TNFalpha, a medium exchange was carried out and cells were either further incubated with the cytokine (TNFalpha without recovery; w/o R) or incubated with normal medium without TNFalpha (TNFalpha with recovery; w/R) for another 48 h. ( A ) TEER values of groups treated with TNFalpha removal were compared to cells treated without removal of the cytokine, respectively (one-way ANOVA, ** p < 0.01; n = 7-13). ( B ) Representative Western blot bands and ( C ) Densitometry of IPEC-J2 cells with or without recovery after 48 h incubation with TNFalpha revealed an increase in claudin-1 and occludin after removal of the cytokine, while claudin-3 expression did not show significant changes. The expression level of TNFR-1 also did not seem to be affected by TNFalpha removal. The shown data are presented as mean +- SEM (one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3).

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Experimental details: Fig. 6 Activated Ezrin inhibits SOCE by directly binding to Orai1 and promotes retraction of membrane blebs. a Representative still images of membrane blebbing in DLD1 cells expressing Lifeact-RFP and GFP-E-Syt1 from five independent experiments. See also Supplementary Movie 6 . b , c DLD1 cells expressing Lifeact-RFP with either GFP-E-Syt1 ( b ) or GCaMP6s ( c ) were treated with the actin polymerization inhibitor Latrunculin B (LatB, 10 uM). Arrowheads show disrupted actin cortex. Results shown are representative of three independent experiments. See also Supplementary Movie 7 . d - f Biochemical analyses of ezrin's role in STIM1-Orai1 complex formation. d Orai1-HA was co-expressed with either GFP or GFP-ezrin. Inputs and HA immunoprecipitates were immunoblotted with antibodies against HA and GFP. e STIM1-FLAG and Orai1-HA were co-expressed with either GFP or GFP-ezrin. Inputs and FLAG immunoprecipitates were immunoblotted with antibodies against FLAG, HA, and GFP. f GFP expression levels (shaded in green) and co-precipitated Orai1-HA (individual measurements) were quantified and normalized to GFP-expressing control. Data presented are means +- SD based on the values from four independent experiments. g , h Proximity ligation assay (PLA) for in situ detection of Orai1-STIM1 interaction. Representative images from five independent experiments are shown in g . h Quantification of PLA signals in expanding (low Lifeact intensity) and retracting (high Lifeact intensity) blebs ba

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Experimental details: Figure 1. Claudin-3 protein levels in CRC samples and survival features of molecular subtypes substratified by CLDN3 expression levels. (A and B) Samples of adjacent normal tissue (N) and tumor tissue (T) were obtained and processed for further analysis of claudin-3 levels by western blotting (n=14). Results of four representative patients are shown. The line graph represents the increase (blue) or decrease (yellow) in the ratio of claudin-3 densitometry units normalized by endogenous protein control (GAPDH) in tumor and normal samples of each patient. Prognostic value of CLDN3 expression (upper and lower tertiles) in (C) unclassified data (n=414) and within (D) CMS1 (n=69), (E) CMS2 (n=250), (F) CMS3 (n=44), and (G) CMS4 (n=52). CRC, colorectal cancer; CLDN3 , gene encoding claudin-3.

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Experimental details: Figure 2. Effects of N -glycan biosynthesis inhibition on TJ stability. (A) Cell lysates from Caco-2, HT-29, and HCT-116 cells were analyzed by western blotting for claudin-3. (B) HCT-116 cells were treated with different concentrations of tunicamycin for 24 h. After treatment, the cells were incubated with FITC-conjugated lectin L-PHA and analyzed by flow cytometry. The histograms of fluorescence were generated by the Cell Quest software: control (purple); 0.25 ug/ml (green); 0.5 ug/ml (pink); 0.75 ug/ml (blue); and 1 ug/ml (orange). (C) Lectin L-PHA specificity. (D) Cell lysates were obtained after 24 h treatment with tunicamycin and analyzed by western blotting for claudin-3, occludin, and ZO-1. Tubulin was used as an endogenous protein control. (E, upper panel) Cell monolayers were fixed and stained for claudin-3 (green) and nuclei (blue) (DAPI). Representative images were obtained by confocal microscopy. The graphs represent the fluorescence intensity in cytosolic and membrane regions of neighboring cells. Scale bar, 10 um. (E, lower panel) Cells were cultured in filters of Transwell polycarbonate, and the functionality of TJs was analyzed by transmission electron microscopy (MET) using ruthenium red as a tracer. The images are representative of ultrathin sections of treated and control cells. Black arrows indicate the cell-cell contact. Scale bar, 2 um. The numerical values represent densitometric units +- standard error (n=3). ns, not significant (P>0.05); *P

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Experimental details: Figure 3. Effects of inhibition of N -glycan biosynthesis on RTK functionality. (A) After treatment with tunicamycin, cell lysates were obtained and analyzed by western blotting for p-EGFR, EGFR, p-IGF1R, and IGF1R. (B and C) Cells were treated with different concentrations of PD153035 or OSI906 for 24 h, and then levels of RTK phosphorylation and claudin-3 were assessed by western blotting. (D) Illustration showing RTK-related or non-RTK-related pathways, as well as on the relationship of this regulatory network with the regulation of CLDN3 expression. (E-H) Effects of specific inhibitors on claudin-3 levels. Cells were treated with different concentrations of (E) U73122 (an inhibitor of PLC-dependent processes), (F) PD98059 (a MEK1 inhibitor), (G) Ly294002 (a PI3K inhibitor), and (H) STA-21 (a STAT3 inhibitor) for 24 h, and then levels of claudin-3 were assessed by western blotting. (I) Cells were treated with paired combinations of inhibitors: PD98059 (21 ug/ml) and Ly294002 (6 ug/ml), or STA-21 (7.5 ug/ml) and U73122 (21 ug/ml). (J) Cells were treated with Forskolin or H-89 (a PKA inhibitor) for 24 h, and then levels of claudin-3 were assessed by western blotting. The numerical values represent densitometric units +- standard error (n=3). ns, not significant (P>0.05); *P

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Experimental details: Figure 6 SiRNA targeting of PVT1 exon 9 induces claudin 4 protein re-expression in MDA MB 231 CL TNBC cells. ( A ) MDA MB 231 CL TNBC cells were transfected with PVT1 exon 9 specific siRNAs (siPVT1 exon 9) for 24 h. Western blotting was performed using specific antibodies against claudin 1, claudin 3, claudin 4, claudin 7 and E-Cadherin. SiRNA targeting of PVT1 exon 9 induced claudin 4 protein re-expression in MDA MB 231 CL TNBC cells in comparison to MDA MB 231 CL TNBC cells transfected with only control scramble non-targeting siRNA (siScramble). ( B ) Quantification of relative claudin 4 protein expression normalized to GAPDH protein expression; N = 2.

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Experimental details: Figure 2 Edoxaban treatment stabilizes the blood-brain barrier and leads to anti-edematous effects in ischemic stroke. ( A ) Left : Representative corresponding coronal brain sections of vehicle (veh)-, edoxaban (Edox)- or phenprocoumon (Phen)-treated mice after the injection of the vascular tracer Evans Blue. Brains were analyzed at day 1 after transient middle cerebral artery occlusion (tMCAO) (Scale bar = 10 mm). Vascular leakage was decreased in the cortical and subcortical areas after edoxaban treatment. Right : Tissue concentration of Evans Blue in the ischemic (ipsilateral) and contralateral hemispheres of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO determined by photometry ( n = 5-8). ( B ) Edema formation as measured by brain water content in the ipsilateral and contralateral hemispheres of vehicle-, edoxaban-, or phenprocoumon-treated mice 24 h after tMCAO ( n = 4-6). ( C ) Top : Claudin (Cldn)-3,-5 and Occludin (Occl) protein expression in the ischemic cortices and basal ganglia of vehicle-, edoxaban-, or phenprocoumon-treated mice at day 1 after tMCAO as determined by immunoblot. Actin was used as loading control. Bottom : Densitometric quantification of Cldn-3, -5 and Occl immunoreactivity in the ipsilateral cortex and basal ganglia of vehicle-, edoxaban-, or phenprocoumon-treated mice ( n = 4). * p < 0.05.

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Experimental details: Figure 2 Localization of claudin-3 ( A --control, B --tumor) and claudin-4 ( C --control, D --tumor) in rat colon by immunofluorescence confocal microscopy (green); bar: 50 mum.

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Experimental details: Fig. 1. A1AT C-terminal peptides induce TJ assembly. ( A ) Schema of recovery from the DSS-induced colitis model in mice. ( B ) Immunofluorescence of claudin-3 and DAPI in colon sections from mice treated as in (A). Scale bar, 20 mum. ( C ) Schema of the CCM treatment of A431 cells. ( D ) Immunofluorescence of claudin-1 and alpha-catenin in A431 cells treated for 3 hours with CCM from mice treated as in (A). Scale bar, 20 mum. ( E ) Quantification of relative claudin-1 intensity at cell-cell boundaries shown in (D) [ n = 5 images (ctrl and recover), n = 6 images (DSS) from two independent samples]. ( F ) Amino acid sequences of the JIP m35 , JIP m36 , and JIP m40 . ( G ) Western blot of CCM with anti-A1AT C terminus antibodies. Quantitative value of each band was presented. ( H ) Quantification of relative claudin-1 intensity at cell-cell boundaries of A431 cells treated with the indicated peptides (20 muM) or HBSS for 3 hours. See fig. S5B ( n = 5 images from two independent samples). ( I ) Relative barrier permeability of A431 cells ( n = 3 independent samples). ( J ) Immuno freeze-fracture replica electron microscopy of A431 cells treated with JIP m35 (20 muM) or HBSS and immunolabeled with anti-claudin-1 antibodies. NM, nuclear membrane; Cyt, cytoplasm; PF, P face. Scale bar, 500 nm. ( K ) Immunofluorescence of claudin-4 and Epsilon-cadherin in EpH4 cells treated with or without JIP m35 (70 muM) for 3 hours. Scale bars, 10 mum. ( L ) TER measurements of EpH4 cells treated

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Experimental details: Fig. 4. Administration of JIPs restores TJs and ameliorates the symptoms of the DSS-induced colitis model in mice. ( A ) Schema of JIP m35 administration to DSS-induced colitis mice. ( B ) Immunofluorescence of claudin-3 and E-cadherin in colon sections from mice treated with DSS or tap water with 2.3 mg/kg per dose JIP m35 , JIP m35-mut1 , or the vehicle control for 10 days. Scale bar, 20 mum. Similar results were obtained in two independent experiments. ( C ) H&E staining of colon sections on day 10. Scale bar, 50 mum. Similar results were obtained in two independent experiments. ( D ) Relative intestinal permeability measured by plasma leakage of FITC-dextran (4 kDa) (ctrl + vehicle, DSS + JIP m35 ; n = 8 mice, ctrl + JIP m35 ; n = 7 mice, DSS + vehicle; n = 6 mice, DSS + JIP m35-mut1 ; n = 4 mice). ( E ) Immunofluorescence of JIP m35 and E-cadherin in colon sections from mice treated with DSS or tap water with 11.1 mg/kg JIP m35 or the vehicle control for 10 consecutive days. Scale bar, 20 mum. Similar results were obtained in two independent experiments. ( F ) DAI in accordance with assessment of stool consistency and fecal blood ( n = 3 mice). ( G ) Quantification of the numbers of Gr-1-positive cells in colon sections from mice treated as described in Fig. 4A ( n = 3 images from three mice). See fig. S11D. ( H ) Body weight changes in mice treated with DSS or tap water with daily administration of JIP m35 , JIP m35-mut1 , or the vehicle control for up to 25 consecutive

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Experimental details: Fig. 5. Human A1AT C-terminal peptides induce TJ assembly in A431 cells and DSS-induced colitis mice. ( A ) Amino acid sequences of the human A1AT C terminus, JIP h42 /CAAP48, and mutant forms of JIP h42 /CAAP48 used in this study. ( B ) Immunofluorescence of claudin-1 and alpha-catenin in A431 cells treated with the indicated peptides at 20 muM or control HBSS for 3 hours. Scale bar, 20 mum. ( C ) Quantification of relative claudin-1 intensity at cell-cell boundaries of A431 cells shown in (B) ( n = 5 images from independent two samples). Similar results were obtained in two independent experiments. ( D ) Relative barrier permeability measured by paracellular tracer flux analysis using FITC-dextran (4 kDa) in A431 cells treated as in (B) and (C) ( n = 3 independent samples). ( E ) Measurement of G 13 activation in the presence of JIP h42 , JIP h42-mut1 , JIP h42-mut2, JIP h42-mut3 , and JIP h42-mut4 , which was assayed in GEF buffer with 5 pmol G 13 , 10 muM GTP, and 10 mM DTT. Synthetic peptides were prepared at the indicated concentrations ( n = 3 independent samples). ( F ) Schema of JIP h42 /CAAP48 administration to DSS-induced colitis mice. ( G ) Immunofluorescence of claudin-3 and E-cadherin in colon sections from mice treated with DSS or tap water with 1.5 mg/kg per dose JIP h42 /CAAP48 or vehicle control for 10 days. Scale bar, 20 mum ( H ) DAI in accordance with assessment of stool consistency and fecal blood ( n = 5 mice). ( I ) Immunofluorescence of Gr-1 in colon

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Experimental details: Figure 2 Expression of CLDNs protein in native and transfected tumor cell lines. Cells were subjected to immunostaining with corresponding antibodies using fluorescein-conjugated secondary antibodies, showing red signals for CLDN-3 and -7 and green signals for CLDN-4. DAPI was used for blue nuclei staining. Images were observed under confocal microscopy. Arrows indicate CLDN localization on cell-cell contact.

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Experimental details: FIGURE 5 Effects of Limosilactobacillus reuteri EFEL6901 strain on tight junction protein levels in mouse colon. (A) Representative western blotting of E-cadherin, occludin, claudin-3, and beta-actin (loading control). Quantification of E-cadherin (B) , occludin (C) , and claudin-3 (D) protein expression levels in the colon. Results are expressed as means +- standard error ( n = 7). Significant differences are presented with the DSS group: * p < 0.05; ** p < 0.01.

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Experimental details: Histological analysis of SMG-ECM-treated cultures reveals that aggregates display an expression pattern different from monolayer cells. A Paraffin sections of cells from 14 day cultures (i.e., untreated BM-MSCs [Untreated], SMG-ECM-treated aggregates [Agg/SMG-ECM], and monolayer [Mono/SMG-ECM] cells) were prepared and stained with H&E or PAS. PAS-positive cells are identified with arrows in the figure. Bar: 50 um. B Immunofluorescence staining of cells prepared as in A and rat SMG tissue (positive control) was used to evaluate the presence and localization of amylase, Aqp5, Mist1, Krt14, Cldn3, and Cldn10 protein. Staining with nonspecific isotype antibody was used as a negative control (not shown). Bar: 50 um

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Experimental details: 2 FIGURE Validation of fluorophore conjugated primary antibodies on LNCaP cells: LNCaP cells were fixed, permeabilized, incubated with 1:100 of each antibody, and imaged with a confocal microscope. Fluorescent signal from primary antibody is shown in first column, nuclear 4',6-diamidino-2-phenylindole (DAPI) stain in second column and merged image in third column. Arrows point to accurate cellular locations of specific fluorescent signal acquired from each antibody staining. Antibodies conjugated to Alexa Fluor 488 (AF488) and Alexa Fluor 647 (AF647) are shown in green and red respectively, DAPI signal is shown in blue. CD9 stains cell membrane, fatty acid synthase (FASN) is found in cytoplasm, fibrinogen is not expressed by LNCaP cells, phospho-histone H2A.X (gammaH2AX) is visualized as nuclear puncta, phosphoglycerate kinase 1 (PGK1) stains the cytoplasm, and claudin 3 (CLDN3) stain is localized to the membrane (scale bars are 20 um).

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Experimental details: Claudins form characteristic copolymers including segregation of channel-forming claudins from Cldn3. a Scheme is illustrating predicted claudin-claudin organization patterns. Two claudins (yellow and magenta) that are compatible with each other tightly colocalize in the TJ (pink). Incompatible claudins separate into different strands or even larger claudin-specific parts. This separation might facilitate permeability of specific ions (cyan) over the TJ meshwork. b Representative STED images of TJ-like meshwork of the observed five organization patterns formed by the indicated co-expressed claudins (SNAP-tagged (yellow; BG-Atto590) and YFP-tagged (magenta; alpha-GFP-NB-Atto647N)) in fixed COS-7 cells are shown. Tight colocalization: intermixing of mesh-forming claudins, integration of non-mesh-forming with a mesh-forming claudin or induction (de novo mesh-forming of co-expressed non-mesh-forming claudins). Separated claudins: segregation and exclusion. c Pearson correlation analysis of Cldn3 co-expressed with barrier-forming (gray) or channel-forming (magenta) claudins. Co-expression of Cldn3 with Cldn3 (yellow) served as positive control. Data represent the mean +- SD. Every n represents the Pearson of one TJ-like meshwork. n (Cldn3 + Cldn3) = 55; n (Cldn3 + Cldn1) = 54; n (Cldn3 + Cldn5) = 20; n (Cldn3 + Cldn6) = 20; n (Cldn3 + Cldn7) = 17; n (Cldn3 + Cldn9) = 15; n (Cldn3 + Cldn14) = 19; n (Cldn3 + Cldn19b) = 25; n (Cldn3 + Cldn2) = 36; n (Cldn3 + Cldn10a) = 30; n (Cldn3 +

Supportive validation

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Experimental details: Segregation enables paracellular ion flux over the TJ meshwork. a Representative confocal images of MDCKII QKO cells stably expressing (sT) FLAG-Cldn2, FLAG-Cldn10a, and FLAG-Cldn2+FLAG-Cldn10a. Single claudin expressing cells were immunostained with anti-Cldn2 (magenta; 2 nd -Atto647N) or anti-Cldn10 (magenta; 2 nd -Atto647N) and anti-ZO-1 (yellow; 2 nd -AF594) antibody. Double claudin expressing cells were immunostained with anti-Cldn2 (magenta; 2 nd -Atto647N) and anti-Cldn10 (yellow; 2 nd -AF594). The white rectangle indicates the region of interest for the magnification in ( b ). b Magnification of FLAG-Cldn2+FLAG-Cldn10a expressing cells from ( a ). White arrows point out differently sized TJ parts that contain only Cldn10a. c Representative confocal images of MDCKII QKO sT FLAG-Cldn3, FLAG-Cldn15 and FLAG-Cldn3+FLAG-Cldn15. Single and double claudin expressing cells were stained with anti-Cldn3 (magenta; 2 nd -Atto647N) or anti-Cldn15 (magenta; 2 nd -Atto647N) and anti-ZO-1 (yellow; 2 nd -AF594) antibody. d Summary of the TER (ohm*cm 2 ), fluorescein flux (10 -6 cm/s) and PNa/PCl ratio from the measured cell lines: MDCKII, MDCKII QKO and MDCKII QKO sT FLAG-tagged Cldn2, Cldn3, Cldn10a, Cldn15 and MDCKII QKO sT FLAG-tagged Cldn2 + Cldn10a and FLAG-tagged Cldn3 + Cldn15. Data represent the mean +- SD from 3 to 5 independent experiments. Single data points are shown in Supplementary Fig. 12 . e Absolute permeability for sodium (PNa; magenta bars) and chloride (PCl; cyan b

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Experimental details: Centella asiatica -derived endothelial epidermal growth factor mitigates radiation-induced enteritis with epithelial barrier restoration in mouse model. Mouse groups are as follows: control (Con), irradiated (IR), irradiated mouse administrated with the conditioned media (CM) of irradiated HUVECs (IR + CM), CM of Centella asiatica (CA)-treated irradiated HUVECs (IR + CA-CM), and recombinant EGF (IR + rEGF). ( A ) Hematoxylin and eosin staining of mouse intestinal tissue sections was performed in each group. ( B ) Proliferation was assessed by staining intestinal sections with ki-67. ( C ) The lengths of villi and crypts in the intestinal sections were quantified. Histological scoring was assessed based on the degree of epithelial architecture maintenance, crypt disruption, vascular enlargement, and infiltration of inflammatory cells in the lamina propria of the ileum of Con, IR, IR + CM, IR + CA-CM, and IR + rEGF groups (0 = none, 1 = mild, 2 = moderate, and 3 = high). ( D ) Body weights of Con, IR, IR + CM, IR + CA-CM, and IR + rEGF groups were determined. ( E ) Immunohistochemistry against epithelial barrier-related molecules, e.g., villin, zonula occuludens 1 (Zo1), desmoglein 2 (Dsg2), and claudin 3 (Cldn3), in the Con, IR, IR + CM, IR + CA-CM, and IR + rEGF groups was performed. ( F ) mRNA levels of the epithelial barrier-related molecules in Con, IR, IR + CM, IR + CA-CM, and IR + rEGF groups were assessed by qRT-PCR. Data are presented as the mean +- standard error of t

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Experimental details: Figure 5 (A) Representative images of immunofluorescence against claudin-3 and claudin-4 in tertiary lymphoid organ-positive and -negative goat mammary gland tissues. Occludin is a marker for tight junctions. Scale bar, 100 mum. (B) Bands detected by western blot and the results of densitometry analyses. Alpha-tubulin serves as internal control; (-) lymphocyte aggregation-negative group; (+) lymphocyte aggregation-positive group. Different letters indicate a significant difference between groups (p < 0.05).

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Experimental details: Supplementary Figure 4 The expression of claudin-3 in IL-22 -/- and WT mice. (A) IL22 -/- , n=6 and WT, n=5 mice uterine samples were harvested 48 hours post intraperitoneal LPS injection. Total mRNA samples were separated and processed by qRT-PCR to evaluate the expression of claudin-3. The Ct values were normalized against the Ct values for GAPDH from the same preparation. The data are provided as mean +- SEM from 3 independent experiments. (B) IL-22 -/- , n=6 and WT, n=5 mice uterine samples (half of one horn) were harvested 48 hours post intraperitoneal LPS injection, frozen fixed, and processed for IHC analysis. (C) H-score of claudin-3 was calculated to measure whether the difference of the staining between the groups is statistically significant. The data from t-test are provided as mean +- SEM. WT, wild type, IL-22 -/- , IL-22 knock out, H-score, histological score. Original magnification 40x, scale bar 100 um. Click here for additional data file.

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Experimental details: FIGURE 2: The localization of some, but not all, TJ proteins to the AJC is attenuated in ZO dKD cell lines. (A-C) A 1-mum-thick, maximum-density projection of the apical domain, en face, acquired at the AJC (see Materials and Methods ). (A) Occludin and cingulin localization at the AJC is reduced, whereas JAM-A and tricellulin appear relatively normal in dKD cells relative to control cells. Claudin-4 staining (green) is included with tricellulin staining (red) to highlight tricellular junctions. (B) Claudin-1 and -2 staining at the AJC is reduced, whereas claudin-3 and -4 appear relatively normal in dKD cells. Note again the dramatic change in cell shape at the AJC in dKD cells. Scale bar: 10 mum.

Supportive validation

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Experimental details: Figure 2 Establishment of claudin-1 (CLDN-1) knockout clones in Caco-2 cells. ( A ) Genomic DNA isolated from wild-type Caco-2 cells or CLDN-1 knockout clones was subjected to PCR for amplification of the CRISPR-Cas9 targeting site in the cldn-1 gene. After purifying and annealing the PCR products, the products were incubated with T7 endonuclease I, which recognizes and cleaves mismatched DNA. Two shorter bands of the predicted size were generated in some clones, indicating that CRISPR-Cas9 had successfully introduced indel mutations at the targeted cldn-1 site. This analysis indicated that the Ko-C1 clone contains only WT alleles, but the Ko-T1 and Ko-C12 clones had deletion alleles. ( B ) Immunoblots comparing CLDN-1 and CLDN-4 production by control Caco-2 cells and CLDN-1 knockout clones. These results indicated that the Ko-C12 clone completely lost the ability to produce CLDN-1, so this clone was selected for further experiments. ( C ) Immunoblot analysis comparing production of other tight junction proteins, including claudin-3 (CLDN-3), claudin-4 (CLDN-4), occludin and ZO-1, by CLDN-1 mutant cells vs. wild-type Caco-2 cells. GAPDH and E-cadherin were used as endogenous controls. For these blots, target proteins in cell lysates (30 ug protein/sample) were detected by Western blotting using specific primary antibodies and suitable horseradish peroxidase-labeled secondary antibodies. Results shown are representative of three repetitions.

Supportive validation

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Experimental details: FIGURE 2: Endogenous clds are variably distributed between the apical tight junction and lateral membrane in MDCK II cells. (A) Top, Maximum-intensity projections of immunofluorescence confocal colocalization of cldn2, cldn1, ZO-1, and merge (left to right panels) show that cldn1 is more laterally disposed than cldn2 or ZO-1. Bottom, Colocalization of cldn4, cldn3, ZO-1, and merge (left to right panels) reveals cldn4 and cldn3 at the lateral membrane as well as colocalized with ZO-1. Arrows in enlargement show apparent concentrations of lateral cldns likely related to lateral membrane infoldings (see Supplemental Figure S1). (B) z -Axis images as in A show ZO-1 focused at the apical junctional region and cldns colocalized with ZO-1 but also distributed along the lateral membrane.

Supportive validation

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Experimental details: Figure 2 Stimulation of the mesencephalic locomotor region does not modulate photothrombosis-driven tight junction protein degradation. ( A , B ), Representative anti-occludin Western blot analysis (cc, cortex contralesional; ci, cortex ipsilesional; bgc, basal ganglia contralesional; bgi, basal ganglia ipsilesional) and densitometric quantification of occludin protein expression in the basal ganglial as well as cortical regions 27 h after photothrombosis of unstimulated (sham) and high-frequency stimulated (stim) rats ( n = 5-7/group). One-way ANOVA. * p < 0.05; ** p < 0.01. ( C , D ), Representative anti-claudin 5 Western blot analysis (cc, cortex contralesional; ci, cortex ipsilesional; bgc, basal ganglia contralesional; bgi, basal ganglia ipsilesional; bd, beyond detection limit) and densitometric quantification of claudin 5 protein expression in the basal ganglial as well as cortical regions 27 h after photothrombosis within the two treatment groups ( n = 5-7/group). One-way ANOVA. ( E , F ), Representative anti-claudin 3 Western blot analysis (cc, cortex contralesional; ci, cortex ipsilesional; bgc, basal ganglia contralesional; bgi, basal ganglia ipsilesional; bd, beyond detection limit) and densitometric quantification of claudin 3 protein expression in the basal ganglial as well as cortical regions 27 h after photothrombosis within the two treatment groups ( n = 5-7/group). One-way ANOVA. *** p < 0.001.

Supportive validation

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Experimental details: Figure 9 Effects of deoxynivalenol (DON), T-2 toxin, and zearalenone (ZEN) exposure on ( A ) occludin (OCLD) and ( B ) claudin (CLDN)-4 localization in BeWo cells, visualized by immunofluorescence staining (400x magnification). Scale bars represent 50 mum.

Supportive validation

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Experimental details: Figure 2. Localization of TJ proteins in claudin quinKO cells. (A-J) Immunofluorescence analyses of parental MDCK II cells (A-J) and claudin quinKO cells (A'-J'). (A-E) Claudin-1 (A), claudin-2 (B), claudin-3 (C), claudin-4 (D), and claudin-7 (E) expression was abolished in claudin quinKO cells. (F) Occludin localization to apical junctions was reduced. (G-I) ZO-1 (G) and ZO-2 (H) were more concentrated at apical junctions, while ZO-3 (I) localization was not altered in claudin quinKO cells. (J) JAM-A was more concentrated at apical junctions in claudin quinKO cells. Graphs are quantitation of the fluorescence intensity and represent mean +- SD ( n = 3 each). *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared by t test. Scale bar: 20 um. n.s., not significant.

Supportive validation

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Experimental details: Figure 3. Cholesterol is enriched in the TJ-containing PM fraction. (A) C1L cells were fixed and stained with an anti-claudin-1 pAb. (B) C1L cells were fixed and stained with an anti-claudin-1 pAb (green) and phalloidin (red). Bars, 10 um. (C) Immunoblot analysis of the PM and IM fractions of C1L cells. Each membrane fraction (5 ug) was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies against the indicated marker proteins (left). Coomassie brilliant blue (CBB) staining is shown on the right. (D) Positive ion mass spectra of SM species in the PM fractions of L and C1L cells. The SM molecular species corresponding with each peak are indicated. The x and y axes show the total carbon chain length and the number of carbon-carbon double bonds of individual lipid molecular species, respectively. (E) Quantification of the indicated SM species in the PM fractions of L cells and C1L cells. (F) Quantification of the cholesterol-to-phospholipid ratio in the PM fractions of L and C1L cells. (G) Immunoblot analysis of the DRM and non-DRM fractions of WT and alpha-catenin-KO EpH4 cells using pAbs against the DRM marker proteins claudin-3 and caveolin-1. Results in C, D, and G are representative of three independent experiments. (H) Quantification of the ratio of the claudin-3 level in the DRM fraction to that in the non-DRM fraction in WT and alpha-catenin-KO EpH4 cells. Error bars show SD calculated based on three independent experi

Supportive validation

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Experimental details: Figure 4 kAE1 protein is in close proximity and interacts with claudin-4. ( A ) Confluent mIMCD3 cells stably expressing kAE1 protein in an inducible manner were grown for 10 days in a 10 cm dish, lysed (Input) and claudin-4 was immunoprecipitated using a rabbit anti-claudin-4 antibody (IP lanes). As a control an irrelevant IgG antibody was used (right panel). Eluted proteins were separated on SDS-PAGE gel prior to immunoblotting with a mouse anti-claudin-4 (bottom blot) or mouse anti-HA (top blot) antibody. Dark circle corresponds to kAE1 carrying complex oligosaccharide, white circle indicates kAE1 carrying high mannose oligosaccharide. ( B ) A similar experiment was performed but immunoprecipitating claudin-3. No kAE1 co-immunoprecipitated with claudin-3 (top panel). ( C ) The claudin-4 immunoprecipitation was repeated using mIMCD3 kAE1 E681Q cells and shows that claudin-4 interacts with the mutant. ( D ) Proximity ligation assay was performed on mIMCD3 cells stably expressing kAE1 grown for 10 days on semi-permeable filters. CNX, corresponding to calnexin, was used as a positive control 15 , Cldn-4 corresponds to claudin-4. Cells were examined under a confocal microscope using a 63 X objective and sections (X-Z) or top views (X-Y) are shown. Red signal indicates that the 2 proteins labeled are within 30 to 40 nm of distance from each other. Nuclear staining is shown in blue as DAPI staining.

Supportive validation

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Experimental details: Fig. 1 Claudin (CLDN) 3. Positive expression in BRCA1 breast cancer (BC) ( a ) and negative control (isotypic antibody) ( b ). Positive CLDN4 expression in BRCA1 -mutated BC ( c ), negative control of CLDN4 (isotypic antibody) ( d ). Positive CLDN7 in BRCA2 BC ( e ), negative CLDN7 in BRCA1 -related tumor ( f ), and negative control of CLDN7 (isotypic antibody) ( g ). Positive CK5 staining in BRCA1 BC ( h )