71-7800

antibody from Invitrogen Antibodies

Targeting: CLDN1 ILVASC, SEMP1

Provider product page for 71-7800

Western blot

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Antibody data

Antibody Data
Antigen structure
References [125]
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Validations
Western blot [1]
Immunohistochemistry [2]
Other assay [81]

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Product number: 71-7800 - Provider product page
Provider: Invitrogen Antibodies
Product name: Claudin 1 Polyclonal Antibody (MH25)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody reacts with the ~ 22 kDa Claudin-1 protein. Reactivity was confirmed by western blotting and immunofluorescence. Positive controls include MDCK cells (dog), Caco-2cells (human), mouse liver, rat liver, and rat kidney.
Antibody clone number: MH25
Concentration: 0.25 mg/mL

CLDN1 protein structure - 71-7800 shown in red.

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Experimental details: Figure 4 Validation of specificity and enrichment of tight junction complexes . (A) Silver stained gel showing steps of purification. Lane 1, whole cell lysate; lane 2, 30,000 x g supernatant; lane 3, 100,000 x g membrane; Lane 3', output 100,000 x g membrane after immuno-isolation, Lane 3"", output 100,000 x g membrane after control immuno-isolation; lane 4, immuno-isolated tight junction complexes; lane 5, control immuno-isolation. MW markers are 200, 116, 96, 66, 45, 38, 25, 14. (b) Western blots of tight junction markers occludin and claudin-1. Lanes 1-5, same as A. (C) Negative staining of complexes showing assemblies of a heterogeneous population of proteins in various sizes. Control immunopurification shows relatively clean background. Scale bars, 100 nm. (D) Higher magnification of tight junction complexes shows globular proteins linked together forming beaded-necklace arrays reminiscent of tight junction seen in vivo (see Results).

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Experimental details: Figure 5 Effects of suppression of cofilin activation or overexpresssion of TJ proteins on the capsaicin-induced cofilin dephosphorylation and decrease in occludin expression. (A-C) Flag-HA tagged cofilin and LIMK, and EGFP-occludin and claudin-1 stable transfectants were established. Monolayers prepared from the transfectants and non-transfected MDCK cells were exposed to 300 uM capsaicin for the time indicated. After lysis, protein expression was analyzed by immunoblotting. Each experiment was performed with at least two different clones and repeated at least twice. (D) MDCK monolayers were pretreated with vehicle or 50 uM TFP for 30 min, and then exposed to 100 uM capsaicin for the time indicated. The levels of occludin and phospho-cofilin were analyzed by western blotting.

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Experimental details: Figure 7 Cofilin activation, decrease in occludin expression and actin alteration in the recovery phase of capsaicin treatment. (A) Western blot detection of phospho-cofilin and occludin in total extracts exposed to 300 uM capsaicin for the times indicated. (B) MDCK monolayers were exposed to 300 uM capsaicin for the time indicated, and then fixed and stained with rhodamine-phalloidin to detect F-actin. Images from each z-section were deconvoluted and overlayed. Bar: 10 um.

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Experimental details: Figure 3 Effects of capsaicin on the distribution of TJ proteins and F-actin. (A, B) Monolayers were exposed to vehicle (A) or 300 uM capsaicin (B) for 45 min and were labeled with each TJ antibody (green), rhodamine-phalloidin (red) and Hoechst (blue). Images were collected as a Z-series, and then deconvoluted and overlayed to display a single composite projection. Top : XY sections of merged images. Bottom : XZ sections of merged images. Scale bar: 10 um. The corresponding TJ antibodies are listed above the images. (C) 3D projection images in a 45deg-angle of claudin-1 and tricellulin staining, plus F-actin. Scale bar: 10 um. (D) MDCK monolayers exposed to vehicle (top) or capsaicin (bottom) as above were stained with E-cadherin. The colors corresponding to each antibody are listed above the images. Scale bar: 2.5 um. (E) Schematic explanation of the protein distributions.

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Experimental details: Figure 3 Nf1 Loss or HRas Activation in the Optic Nerve Causes Changes in Claudins and BBB Permeability (A) Electron micrographs of optic-nerve cross-sections of capillaries 1 mm from the chiasm (50,000x; scale bar, 200nm) show electron-dense TJs between endothelial cells. Blue arrowheads: areas of TJ disruption. (B) Quantification of the total TJs with gaps, with 200-300 TJs counted per genotype. (C) Evans blue stain of longitudinal sections of PLPCre; Nf1fl / fl and CNP-HRas optic nerve. Insets: cross-sections; arrows: blood vessel. (D) Western blots from total optic-nerve lysates showing claudin-1 and claudin-5. All experiments included three to five animals per genotype. *p < 0.001. See also Figure S5 .

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Experimental details: Figure 6 Cx43 mutant-expressing REKs exhibited reduced transepithelial resistance when compared with Cx43 overexpressing REKs in the absence of changes in the expression or localization of junctional proteins ( A ) Transepithelial resistance measurements of confluent monolayer cultures were taken daily for 5 days and the resistance from days 2-4 were averaged and plotted. ( B ) E-cadherin, claudin1 and occludin were localized by immunofluorescent labelling in REKs overexpressing GFP-tagged Cx43, G138R or fs260. Western blot analysis did not reveal any changes in the expression of occludin, E-cadherin or claudin-1 ( C and D ). * P

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Experimental details: Figure 4 Capsaicin decreases occludin protein content and the protein-protein interactions involving occludin in TJ. (A,B) MDCK monolayers were exposed to ethanol control or 300 uM capsaicin for 45 min. (A) The cellular localization of the TJ proteins occludin and claudin-1 was examined by immunofluorescence. Scale bar: 10 um. (B) Western blot detection of occludin, tricellulin, Zo-1, claudin-1, E-cadherin and actin in the cytosol, membrane fractions and total cell extracts. The densitometic analysis of total proteins from three independent experiments performed with NIH ImageJ software. **represents p

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Experimental details: Figure 4 Changes in Cldn1 expression in skin tumorigenesis . In the normal (not shown) and vehicle-treated ( a , after 12 weeks) epidermis, Cldn1 is localized in the basal and suprabasal layers; however in response to the two-stage chemical carcinogenesis protocol, the number of Cldn1-positive epithelial cells was progressively reduced starting from the basal layer and moving upwards at 2 ( b ), 4 ( c ), 8 ( d ) and 12 ( e ) weeks. Although a distinctly membranous Cldn1 association was maintained in the upper layers of the treated epidermis, as the number of Cldn1-negative epidermal cells in the lower epidermal layers increased, only sporadic Cldn1 localization was evident ( c-e ). The epidermal basal layer is indicated by a dotted line, and the suprabasal compartment is marked with a bracket ( a-c ); note that the basal layer is out of view in panels d and e ; the entire view is therefore the suprabasal compartment and is marked with a double-ended arrow.

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Experimental details: Fig. 2. Paracellular permeability of the cultured corneal epithelia was examined by use of the low molecular weight tracer, EZ-Link sulfo-NHS-LC-biotin. The tracer did not penetrate the epithelial sheet neither 1 day after confluence (7 days in cell culture) ( A-D ), nor in 7 day confluent epithelia ( E-H ), as indicated by the (C,G) lack of staining of biotinylated proteins at cell-cell boundaries in the lower layers of the epithelium. (D,H) Maximal projections of the merged channels, transverse optical sections of cultures stained for cldn-1 to immunolocalize TJ (green channel; B,D,F,H). Proteins biotinylated with the tracer are stained in red (C,D,G,H). (A,E) show the aspect of cldn-1 in xyz maximal projections of the stained cell cultures. Dashed squares show the fields examined in the transverse sections. Dashed lines indicate the basal side of the cultured epithelia. In A,E, Bar = 40 um; in B-D, Bar = 16 um; in F-H, Bar = 20 um.

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Experimental details: Fig. 3. TJ components detected in cultured RCE1(5T5) corneal epithelial cells. This was performed either by: ( A-J ) immunostaining, ( K-M ) end point RT-PCR, or ( N ) Western blot. (A) We used alpha-catenin as an indicator of the presence of adhesion complexes in the cultured epithelia. (B,G) Ocln, (D) ZO-1, (E) cldn-1, (C,I) cldn-4, and (F,H) cgn were easily located in cell borders of one day post-confluent cultures. (G-I) are transverse sections showing that TJs (arrows) were located at the cell boundaries of suprabasal cells. In (G), dashed line indicates the boundaries between a suprabasal cell and the basal cell layer; in (H-J) the dashed line indicate the basal side of the epithelium. Nuclei were stained either with propidium iodide or TO-PRO(r)-3. In 7-day confluent epithelia we found (K) the expression of cldn-1, -2, -4, but not of cldn-3 and -7; (L) cgn, ocln, and (M) ZO-1. Primers for cldn-3 and -7 were verified in PCR reactions using mouse kidney cDNA (K, m-kidney) that led to amplification products of the expected size and sequence. PR-P0 was used as an internal marker that does not change during the differentiation process. The antibodies were only appropriate to immunodetect cldn-1 and -4 and cgn (N); lanes 2 and 3 correspond to the loading control for the experiment. PCR reaction in the presence (+) or the absence of cDNA (-) (negative control). Results correspond to six duplicated experiments. A-D: Bar = 20 mum; E,F: Bar = 40 mum; G: Bar = 8 mum, and H-J: Bar

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Experimental details: Fig. 5. Epidermal growth factor modulates the permeability of the corneal epithelial TJ. ( A ) Shows that the cldn-1 immunolocalization pattern is strong with punctuated discontinuities in cell cultures maintained in basal medium without EGF, while ( B ) cells cultured in the presence of 10 ng/ml EGF had a well-defined cell-cell contacts and a typical cldn-1 immunolocalization, with some discontinuities. The highest expression of cldn-1 correlated with ( C ) high TER values in the EGF-supplemented cultures which showed the tightest epithelial barrier. Effect of EGF was significant ( P

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Experimental details: Fig. 6. TJ assembly is disrupted by retinoic acid (RA). In contrast with control cultures ( A ), corneal epithelial cells grown in the presence of RA, had a weak, discontinuous, punctuated pattern of cldn-1 at cell boundaries, which decreased in a concentration-dependent manner; and with cldn immunolocalization in cell cytoplasm in the RA-treated cells ( B-D ). ( E ) This change correlated with a partial (0.1 uM RA) or a complete blockage of the increase of TER observed in control cultures which were not treated with RA. ( P

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Experimental details: Figure 6 Overexpresssion of cofilin, LIMK and occludin, but not claudin-1, significantly diminishes the capsaicin-induced decrease in TER. (A,B) Transfectant monolayers were prepared in transwell inserts, exposed to 300 uM capsaicin, and subjected to TER measurements. Values represent mean +- S.D. Asterisks indicated significant difference from Vector control at the same time point; *, p

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Experimental details: Fig 2 Confirmation of cotransin sensitivity and cotransin resistance of selected proteins using SDS-PAGE/ immunoblotting. After transfection and treatment with cotransin (17 h, 30 muM) (+) or with DMSO (-) secretory and integral membrane proteins were isolated from HepG2 cells. The proteins were identified by SDS-PAGE/immunoblotting using specific primary antibodies and horseradish peroxidise-conjugated anti-mouse or anti-rabbit IgG as secondary antibodies. The cytosolic GAPDH protein does not contain a signal sequence and served as a control for a non-sensitive protein. As examples for secretory proteins (all SPs) cotransin-sensitive Apo B-100 and cotransin-resistant PAI-1 were used. For membrane protein possessing SPs, cotransin-sensitive CDH2 and cotransin-resistant CNX were analyzed. As examples for membrane proteins containing SASs, cotransin-sensitive Erlin2 and cotransin-resistant CLDN1 are shown. The immunoblots are representative of three independent experiments. The bar graphs shown at the right side of each immunoblot represent mean intensities of the respective protein bands of these three independent experiments +-SD (densitometric analysis using ImageJ).

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Experimental details: Figure 5 SRGN elicits NSCLC aggressiveness mediated through Claudin-1 expression. ( a ) Cells were cultured in serum-free medium for 48 h, and western blotting was performed using anti-Vimentin (IF01, Calbiochem, Billerica, MA, USA) and anti-CLDN1 (71-7800, Invitrogen, Carlsbad, CA, USA). ( b ) H1299 cells were transiently transfected without or with vectors encoding GFP or CLDN1. Western blot analysis of designated proteins is shown. ( c ) H1299/SRGN cells stably harboring scramble-shRNA or CLDN1-shRNAs were subjected to western blot analysis of designated proteins. (d) Migration assay of H1299/SRGN cells stably harboring scramble-shRNA or CLDN1-shRNA is shown. Data are presented as the mean+-s.d. of three independent experiments. * P

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Experimental details: Figure 6 SRGN/CD44 axis induces CLDN1 expression via NF-kappaB activation. ( a ) NF-kappaB reporter assay was performed in H1299/Mock and H1299/SRGN cells. ( b ) Cells were cultured in serum-free medium for 48 h, and subjected to western blot analysis using anti-IkappaBalpha (#4812, Cell Signaling Technology) and anti-p65 (sc-372, Santa Cruz Biotech). ( c ) H1299/SRGN cells were transiently transfected with vectors encoding dominant-negative IkappaB kinase alpha (DN-IKKalpha) or DN-IKKbeta and cultured in serum-free medium for 48 h, followed by western blot analysis of IkappaB and CLDN1. HA-fused DN-IKKs were detected by anti-hemagglutinin (sc-805, Santa Cruz Biotech). ( d ) Unsorted H1299 and CD44(-) cells stably harboring the Mock-control or SRGN-expressing vectors were cultured in serum-free medium for 48 h. Nuclear and cytosolic fractions were prepared for western blot analysis using anti-p65, anti-PARP (sc-7150, Santa Cruz Biotech) and anti-tubulin (GTX112141, GeneTex). ( e ) Cells described in d were cultured in serum-free medium for 48 h, and subjected to western blotting of CD44 and CLDN1. ( f ) H1299 CD44(-) cells stably harboring Mock-control or CD44s-expressing vectors were incubated in medium supplemented with CM collected from H1299/Mock or H1299/SRGN cells for 24 h, and subjected to western blot analysis of CLDN1. Data are presented as the mean+-s.d. of three independent experiments. ** P

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Experimental details: Figure 6 Knockdown mIMCD3 cells share molecular and cellular phenotypes. ( a ) Total cell lysates from mIMCD3 cell lines were analysed by western blot with antibodies against collagen IV, E-cadherin and beta-actin. Uncropped western blots are shown in Supplementary Fig. 13a . ( b ) mIMCD3 cell lines were grown on transwell supports for 2 weeks and immunostained with anti-collagen IV antibody. Figures show representative images of three independent experiments of n =3 views of multiple cells each. Scale bars, 10 mum. ( a , b ) Repeated three times. Representative images are shown. ( c ) mIMCD3 cell lines cultured for 3 days in 3D collagen I gels, immunostained with anti-E-Cadherin and anti-Claudin-1 antibodies. Scale bars, 10 mum. ( d ) EM of 3D cultured mIMCD3 cells (top panels), scale bars, 20 mum. Higher magnification of the areas of abnormal extracellular matrix deposits in kd cells (bottom panels). Representative images of n =9 (Control shRNA), n =7 (VIPAR shRNA), n =9 (VPS33B shRNA) and n =8 (LH3 shRNA) spheres are shown. Scale bars, 1 mum. ( e ) LC-MS-MS analysis for the relative quantification of the degree of Lys-O-GalGlc, Lys-O-Gal and Lys-OH in collagen IV from mIMCD3 cell lines. Two different control lines (wt and Control shRNA) and two different VIPAR shRNA clones were used. LH3 shRNA cells were used as a positive control. ( f ) Differentially expressed genes in kd mIMCD3 cell lines were detected using a moderated t -test implemented in the limma package in R 34 .

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Experimental details: Fig. 7 a Heatmap representing the expression values of mucin genes in colonocytes ( Muc ). Each row corresponds to one Muc gene. Each column corresponds to a single rat fed a normal-protein diet (NPD), or a high-protein diet (HPD). The color indicates the relative expression value (as indicated by the key) obtained from microarray experiment and normalized to the mean value in the NPD group. Mean expression values of the HPD and the NPD groups were compared with a t test. *: p < 0.05. b Relative expression values of defensin genes significantly differentially expressed (q < 0.01) in the colonocytes of rats fed a HPD when compared to a NPD. The expression values were obtained by microarray experiment and normalized to the mean value in the NPD group. Defb3, 4, 10, 15, 19, 22 and 30 (beta-defensin 3, 4, 10, 15, 19, 22 and 30). c Claudin 1 protein expression was assessed by western blot in colonocytes of rats fed an HPD or an NPD. Band intensity was quantified and normalized to the intensity of the band corresponding to actin. e - f Barrier function was evaluated with Ussing-chambers in distal colon of rats fed an NPD or an HDP. e Transmural resistance was measured for 15 min after mucosa mounting in the chamber. f FITC-dextran (FD4) transport from mucosal to serosal side was recorded during two hours and FD4 apparent permeability (FD4 P app ) was calculated. c - e For each parameter, mean values were compared with a t test. b - e Data presented on histograms are means +/- S.E.M

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Experimental details: Figure 2 Both EpCAM and TROP2 regulate claudin expression in keratinocytes. HaCaT cells were transfected with control or EpCAM siRNAs ( A ), control, EpCAM, TROP2, or EpCAM and TROP2 siRNAs ( B ) using electroporation. 72 h after transfection, cell lysates were prepared, resolved using SDS-PAGE and immunoblotted with anti-EpCAM, anti-TROP2, anti-claudin-7 or anti-claudin-1 to assess corresponding protein levels. beta-actin was used as a loading control.

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Experimental details: Figure 5 HAI-1 and HAI-2 regulate EpCAM and TROP2 cleavage and claudin levels. HaCaT cells were transfected using electroporation with control siRNA or one of two different SPINT1 siRNAs (siSPINT1-1 and siSPINT1-2) ( A ), control, SPINT1, SPINT2, or SPINT1, and SPINT2 siRNAs ( B ). After 72 h, cell lysates were prepared, subjected to electrophoresis and analyzed via Western blotting for HA1-1, HAI-2, matriptase, EpCAM, TROP2, claudin-1, claudin-7 and E-cadherin. beta-actin was used as a loading control. Representative data from 1 of 3-4 experiments is shown.

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Experimental details: Figure 6 HAIs act via matriptase to regulate degradation of EpCAM, TROP2, and claudins in lysosomes. HaCaT cells were transfected using electroporation with control or SPINT1 plus SPINT2, matriptase, or SPINT1, SPINT2 plus matriptase siRNAs ( A ), or control or SPINT1 and SPINT2 siRNAs ( B ). Before being harvested at 72 h after transfection ( A , B ), cells were treated with or without 100 muM chloroquine for 20 h ( B ). RIPA cell lysates were resolved using SDS-PAGE and immunoblotted with anti-HAI-1, anti-HAI-2, anti-matriptase, anti-EpCAM anti-TROP2, anti-claudin-1, or anti-claudin-7. beta-actin was used as a loading control. Representative data from 1 of 5 experiments is shown. ( C ) Band intensities corresponding to full-length (FL) EpCAM, FL TROP2, claudin-1 and claudin-7 (left panel) and cleaved EpCAM and cleaved TROP2 (right panel) were quantified and normalized to beta-actin. Data are plotted as ratios (means +- SEM) relative to corresponding untreated siControls ( n = 5). A two-way ANOVA was used to calculate p values for multiple group comparisons to assess mean differences between groups (* p < 0.05, ** p < 0.01, *** p < 0.001 or 0.0001, n.s. p > 0.05).

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Experimental details: Figure 7 Western blot analysis of the expression ZO-1 and claudin-1 in DSS-induced colitis mice. Data represent the mean +- standard deviation ( n = 6 mice per group). Tukey's multiple comparison test was performed, and different lowercase letters indicate statistically significant differences ( p < 0.05).

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Experimental details: Figure 3 Delta oprC infection decreased mouse mortality and lung damage following P. aeruginosa infection. (A) C57BL6 mice were intranasally challenged with wild type PAO1, Delta oprC , and complemented strain at 2 x 10 7 CFU in 25 muL PBS; moribund mice were killed to obtain survival data ( n = 6). (B) Bacterial burdens in the BALF were determined 24 h after bacterial infection ( n = 6). (C) Representative images of immunofluorescence staining of the lungs infected with bacteria for Claudin-1 co-stained with ZO-1 and DAPI ( n = 3). Arrowheads show the membrane localization of Claudin-1. Scale bars, 20 mum. (D) Representative histological views of the lungs of mice 24 h after intranasal infection with bacteria by H&E staining (insets show the enlarged views) ( n = 3). Arrows show examples of neutrophil infiltration areas. Scale bars, 50 mum. (E) Quantification of leukocytes, macrophages, monocytes, and neutrophils numbers per 30,000 cells collected from lung infected with bacteria for 24 h. Error bars represent the mean +- s.d. Kolmogorov-Smirnov test was performed for survival curves analysis. One-way ANOVA with a post-hoc Tukey test was performed for comparison of means of groups in other cases.

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Experimental details: Fig. 4 Analysis reveals that the THS model promoted function through tension homoeostasis. a , c Real-time PCR analysis of dermal ECM proteins and epithelial-mesenchymal interaction-related genes ( COL1A1 , COL1A2 , FBN1 , ELN , MMP1 and KGF ) of HSEs. *P < 0.001, as assessed by Dunnett's test following two-way analysis of variance (ANOVA; P < 0.001); error bars represent the standard deviation. ( b ) Immunohistochemical analyses of epidermal and dermal morphogenesis markers in skin equivalents. These samples were stained with COL1, COL7, COL17, CK5, CK10, CLDN1, FLG and Ki67 antibodies. The dotted lines indicated boundary between epidermis and dermis. Scale bars, 50 um. d Analysis of the Ki67-positive basal keratinocytes in skin equivalents. Immunohistochemical analyses (left) and Ki67-positive cell ratio (right) were shown. Scale bars, 20 um. *P < 0.01 and **P < 0.001, as assessed by Dunnett's test following two-way analysis of variance (ANOVA; P < 0.001). e Schematic representation of the methods used for evaluating molecular activity. Functional molecules were applied to skin equivalents by dissolving in medium (as shown by IAM) or lotion (as shown by TAM) and treating for 6 h to 5 days. f The reactivity to ATRA in skin equivalents (Bell's model, THS model, and TRS model) was evaluated by mRNA expression levels of HAS3 and COL1A1 in the IAM assay model. *P < 0.01 and * *P < 0.001, as assessed by Tukey-Kramer test following two-way analysis of variance (ANOVA; P < 0.001);

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Experimental details: Fig. 1 Intestinal barrier was destroyed in NEC patients and mice. a Representative sections from ileac tissue of Ctrl or NEC patients stained with anti-claudin-1, anti-occludin, and anti-ZO-1 antibodies. Neonatal intestinal atresia patients were used as controls. b Statistical analysis of the average optical density of immunohistochemical staining images of ( a ) (n = 5 per group). c and d Western blot analysis of claudin-1, occludin, and ZO-1 protein levels in small intestinal epithelial cells of NEC mice and relative gray calculation (n = 5 ~ 6 per group, representative data from four independent experiments). e The concentration of FITC-dextran 4 kDa (FD4) in the serum of Ctrl or NEC mice. Ctrl, dam-fed, n = 5 ~ 6 per group. Four independent experiments were performed. Data are shown as mean values +- SD. Statistical analyses were performed with Student's two-tailed unpaired t -test. * p < 0.05; ** p < 0.01; n.s., not significant. Scale bars represent 20 mum in ( a )

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Experimental details: Fig. 6 PXR protected barrier function from LPS damage. a TEER changes in Caco-2 cell monolayers treated with LPS for 24 or 48 h. b and c Western blot analysis of claudin-1, occludin, and ZO-1 protein levels in Caco-2 cells treated with LPS (n = 4 per group, representative data from three independent experiments). d Western blot analysis of PXR in Caco-2 treated with LPS. e Confirmation of PXR overexpression by Western blot. f and g Western blot analysis of the components of tight junctions (Claudin-1, Occludin, and ZO-1) in Caco-2 cells treated with LPS for 48 h (n = 4 per group, representative data from three independent experiments). h FD4 flux of basolateral medium was measured after Caco-2 cells were treated with LPS or not treated for 48 h (n = 5 per group, representative data from four independent experiments). Data are expressed as mean values +- SD. Statistical analyses were performed with Student's two-tailed unpaired t -test in ( c and g ), * p < 0.05; ** p < 0.01; n.s., not significant; TEER, transepithelial electrical resistance; FD4, FITC-dextran 4 kDa

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Experimental details: 4 Figure Betaine increases tight junction protein expression in human organ-cultured skin. (a) Claudin-1, claudin-4 and occludin immunofluorescence in ex vivo human skin cultured for 72 h under control conditions, in the presence of 35 mmol L -1 betaine, 10 mumol L -1 N -(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide ( BPDBA ), and both 10 mumol L -1 BPDBA and 35 mmol L -1 betaine. (b) Quantification of fluorescence intensity showed that the presence of BPDBA mitigated the increase in expression of all three tight junction proteins caused by betaine. (c) Claudin-1, claudin-4 and occludin gene expression was not affected by the presence of betaine. Data are presented as the mean +- SEM , two-way anova , n = 7. Bars = 50 mum. SC , stratum corneum.

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Experimental details: 5 Figure Taurine increases tight junction protein expression in human organ-cultured skin. (a) Claudin-1, claudin-4 and occludin immunofluorescence in ex vivo human skin cultured for 72 h under control conditions, in the presence of 35 mmol L -1 taurine and following the addition of 0*1 mumol L -1 ciclosporin A (CsA) and 35 mmol L -1 taurine. (b) Quantification of fluorescence intensity showed that the presence of CsA mitigated the increase in expression of all three tight junction proteins caused by betaine. (c) Claudin-1, claudin-4 and occludin gene expression was similarly affected by the presence of taurine. Data are presented as the mean +- SEM , two-way anova , n = 7. Bars = 50 mum. SC , stratum corneum.

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Experimental details: 7 Figure Immunoblotting of tight junction proteins in human primary keratinocytes. (a) Immunoblotting analysis showed that the expression of claudin-1 and claudin-4 was induced in differentiated normal human epidermal keratinocytes incubated with 5 mmol L -1 organic osmolytes. (b) Densitometric quantification showing the ratio of tight junction protein to beta-actin protein expression. Data are presented as the mean +- SEM , two-way anova , n = 3.

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Experimental details: Figure 4. ( A ) Pretreatment with 25 uM PKC zeta pseudosubstrate inhibitor (PSI) significantly prevented the increase in TEER induced by PGN. All TEER values are expressed as percentage relative to control (the baseline value was > 520 Omega*cm 2 ). Values represent mean +- SD * P < 0.05, ** P < 0.01, *** P < 0.001. The data were analyzed using one-way analysis of variance (ANOVA). All the experiments were performed atleast 3 times during different days with number of replicates (n = 4). Western blotting ( B ) for claudin-1, occludin and ZO-1. Upregulation of claudin-1 and ZO-1 was observed in Calu-3 cells after treatment with P3C and PGN. Occludin was decreased. The images are representative of 3 independent experiments. beta-actin was used as a lane loading control. The number of replicates is (n = 2). ( C ) the corresponding expression levels of claudin-1, occluding and ZO-1 are shown as bar graphs. ( D ) western blotting for claudin-1. The PSI inhibited the claudin-1 expression induced by PGN treatment in a concentration dependent manner (25 uM, 10 uM and 1 uM). E, the corresponding expression levels of claudin-1 are shown as bar graphs. ( F ) western blotting for phospho-PKC zeta in Calu-3 cell monolayers treated with PGN. Stimulation by PGN increased phosphorylation of PKC zeta in the treated cells which was inhibited by pretreatment with PSI 25 uM and 10 muM. Expression of phospho-PKC zeta and total PKC zeta was examined using western-blot analyses of total cell lysate

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Experimental details: 10.1371/journal.pone.0168346.g001 Fig 1 Urine cells on cytospin slides stained for podocalyxin (PCX, red), claudin-1 (CL1, green), and Dapi (blue). (A-D) A Faby podocyte which is PCX+/CL1- and shows characteristic vacuolated cytoplasm. (E-H) A urine cells which is PCX+/CL1+ consistent with a parietal epithelial cell (PEC) phenotype. (I-L) An apoptotic podocyte (PCX+/CL1-) in the urine from a Fabry patient with shrunken cytoplasm and small nucleus (arrow) compared to its adjacent cells. (M-P) A podocyte (PCX+/CL1-) in the urine from a healthy subject does not show vacuolated cytoplasm.

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Experimental details: Figure 1. Immunophenotype of perineurial cells, arachnoid, and suprachoroidalcells. Immunohistochemical profile of perineurial cells (panels A-E),optic nerve sheath/arachnoid (F-J), and suprachoroidal cells (K-O,indicated by black arrows). Panel A shows ciliary nerves and sclera ofthe posterior eye with an inset showing endoneurium (endon.),perineurium (perin.), and epineurium (epin.) of a ciliary nerve.Perineurial cells are immunoreactive for 2 EMA antibodies (B, C), Glut-1(D), and claudin-1 (E). Panel F shows optic nerve sheath with arachnoid,which is immunoreactive for both EMA antibodies (G, H), weakly reactivefor claudin-1 (J), and negative for Glut-1 (I). Panel K shows retina,choroid, thin suprachoroidal cells, and sclera. Suprachoroidal cells,indicated by black arrows, are immunoreactive for EMA (L, M), and, inthese examples, immunonegative for Glut-1 (N) and claudin-1 (O).Examples are from multiple samples with panels B-E and L-O at 600x(panel E, O scale bars = 20 um) and panels G-J at 400x (panel J scalebar = 20 um). Lower-power, PAS-stained images (A, F, K) at 100x.Abbreviations: EMA, epithelial membrane antigen; PAS, periodicacid-Schiff.

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Experimental details: Figure 2. A heat map of staining intensity of suprachoroidal fibroblasts for EMAantibodies (CellMarque, Ventana), CD34, claudin-1, and Glut-1 in theposterior pole, equator, and anterior pole of the eye. Each antibody hasspecimens 1-8 listed in columns with anatomic region of interest listedin rows. A scale for staining intensity is present at the right of thefigure. Abbreviation: EMA, epithelial membrane antigen.

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Experimental details: Figure 4 Increase in plasma membrane surface localization of the D97S mutant by endocytosis inhibitor or primaquine treatment. ( A , B ) MDCK cells stably expressing the FLAG-tagged D97S mutant were treated with MDC and MbetaCD for 3 h. After collecting cell lysates, the aliquots were blotted with anti-FLAG and anti-beta-actin antibodies. The band densities of proteins are represented relative to the values with 0 muM. ( B ) The cells were stained with anti-FLAG (CLDN16) and anti-ZO-1 antibodies. ( C , D ) MDCK cells stably expressing the FLAG-tagged D97S mutant were incubated with primaquine (PQ, 100 muM). The biotinylated proteins were blotted with anti-FLAG and anti-CLDN1 antibodies. ( E ) The cell lysates of MDCK cells expressing the WT or D97S mutant were blotted with anti-FLAG and anti-beta-actin antibodies. The band densities of proteins are represented relative to the values at 0 h. The full-length blot images are shown in Supplementary Figs S6 - S8 . ( F ) The cells were incubated with anti-FLAG (CLDN16) and anti-ZO-1 antibodies. The scale bar indicates 10 mum. n = 4 in four independent experiments. ** P < 0.01 and * P < 0.05 compared with 0 muM.

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Experimental details: Fig. 3 AC mitigates HFD-mediated decrease in TJ protein expression and modulates hormones that regulate TJ structure and function. Mice were fed a control diet (empty bars), the control diet supplemented with 40 mg AC/kg body weight (dashed bars), a HFD (black bars), or the HFD supplemented with 40 mg AC /kg body weight (blue bars) for 14 weeks. A- Ileum protein levels of occludin, ZO-1 and claudin-1 were measured by Western blot. Bands were quantified and values normalized to HSC70 levels (loading control). Values were referred to those of the control group (empty bars). B - Plasma GLP-2 concentrations were measured by ELISA. Results are shown as mean +- SE of 6-8 animals/treatment. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3

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Experimental details: Fig. 3 Cystine (Cys2) improved the decrease of the ratio of claudin-4 in the detergent-insoluble fractions (IS) to soluble fractions (S). Caco-2 cells were separated the detergent-insoluble fractions and soluble fractions after 2 h of incubation with 0.5 mM H 2 O 2 . a Whole cell extracts, detergent-soluble fractions and insoluble fractions of Caco-2 cells were immunoblotted for ZO-1, occludin, claudin-1, claudin-4, and GAPDH. The expression level of the TJ proteins zonula occludens-1 (ZO-1) ( b ), occludin ( c ), claudin-1 ( d ), and claudin-4 ( e ) in the whole cell extracts was measured and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions was calculated. Control cells (Con) were monolayers pretreated with DMEM and not incubated with 0.5 mM H 2 O 2 . Protein expression was normalized to GAPDH levels and expressed as mean fold change relative to the Con group. Values are represented as mean +- SEM ( n = 6)

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Experimental details: Figure 4 Fenofibrate upregulates expression of tight junctions on intestinal epithelial cells. Immunofluorescent staining of claudin-1 (CLD-1) expression in the ( a ) duodenum, ( b ) ileum, and ( c ) colon of diabetic dogs pre- and post-fenofibrate administration. ( d - f ) Semi-quantitation of CLD-1 protein expression in the intestinal compartments. ( g ) Representative ZO-1 staining and ( h ) quantification of barrier tortuosity in Caco-2 cells treated with different concentrations of glucose + 10 muM fenofibrate + 3 muM GW6741 or ( i , j ) TNF-alpha + fenofibrate + GW6741 for 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001 by ( a - f ) mixed models ANOVA and ( g - j ) one-way ANOVA with Tukey's multiple comparison test. N.S., not significant.

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Experimental details: Claudin1 immunohistochemistry in the distal colon. Representative immunohistological images are displayed below at 20X objective. All DiNP treatment groups were compared to control. Quantification of CLDN1 is in the graph on the right. The data are presented as means +- standard error of the mean (SEM). n = 4-6 samples/group.

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Experimental details: Eugenol improved the expressions of the duodenal tight junction proteins ZO-1, claudin-1 and occludin in Salmonella -infected broilers. (A) WB analysis: the protein bands of critical proteins. (B) WB analysis: the quantification of the protein levels by determining the band intensity and normalized to beta-actin band levels. (C) The mRNA expressions of tight junctions (ZO-1, claudin-1 and claudin-2). All experiments were repeated in triplicates. The data are expressed as the Mean +- SE (n = 15). ** P < 0.01, *** P < 0.001 vs. Control. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. SL1344 infection. Figure 5

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Experimental details: Eugenol improved the expression of tight junction protein claudin-1 in the duodenum of Salmonella -infected broilers by immunofluorescence. Green fluorescence represents claudin-1, and blue (DAPI dye) represents the nuclei. Figure 6

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Experimental details: FIGURE 5. A, Representative immunoblots of colon lysates from untreated (left panel) and treated (7 days DSS and 7 days recovery, 14 days, right panel) &plus&sol&plus and fr CR &solfr CR rats with ZO-1, occludin, claudin-1, and actin. Protein quantification of corresponding immunoblots for untreated (B-D) and treated (14 days, E-G) animals for (B, E) ZO-1, (C, F) occludin, and (D, G) claudin-1; n = 6 for each genotype. Actin was used as loading control. H, Short circuit current (I sc ) and (I) transepidermal resistance (TER) in proximal and distal colon of &plus&sol&plus and fr CR &solfr CR animals (untreated, n = 3 per genotype) or following 7 days of DSS treatment and 7 days of recovery (14 days, n = 3 per genotype). J, Diarrhea score in untreated &plus&sol&plus , fr CR &sol&plus , and fr CR &solfr CR animals (n = 4 per genotype). &ast P < 0.05.

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Experimental details: Fig. 7 D88 mitigates the morphologic alterations of the intestinal lining and attenuates intestinal inflammation. a Representative confocal image ( n = 2) of distal ileum with Claudin-1 immunofluorescence staining. The experiment was repeated independently two times with similar results. Samples for confocal imaging were harvested 22 h post-burn and infection. Green fluorescence represents Claudin-1, and blue fluorescence represents the DAPI stain, the white line represents the scale bar (100 um). b Levels of tumor necrosis factor (TNF-alpha) in the distal ileum 22 h post-burn and infection. The total protein was isolated from the distal ileum, and the concentration of TNF-alpha in the sample was quantified using ELISA. c The levels of interleukin (IL-6) in the distal ileum were also quantified using ELISA. b , c Data represent at least n = 5, each dot in the bars represents data from one mouse. The error bars denote +- SEM. One-way ANOVA followed by Tukey post-test was applied. The no-treatment group data (Burn + PA14) were compared to the vehicle-treated and the D88-treated groups. *, **, and *** indicates significant differences compared to the control at P < 0.05, P < 0.01, and P < 0.001, respectively. ns represents no significant difference.

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Experimental details: Figure 4 Constitutive expression of EspF and Map depletes tight junction proteins. ( A ) Cell lysates derived from untransfected, EGFP, EGFP-Tir, EGFP-EspF and EGFP-Map cell lines were separated by electrophoresis on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Equal amounts of cell lysates were loaded after normalizing with GAPDH. Blots were probed with the indicated primary antibodies and HRP-conjugated secondary antibodies. Full-length blots are presented in Supplementary Figure S3 . ( B , C ) The x-ray films were scanned and quantitative analysis was performed by measuring band intensities using ImageJ software. The expression of different tight junction proteins in these cell lines was normalized with respect to untransfected cells and fold changes were calculated. Data are represented as means +- s.e.m. from six independent experiments using two biological replicates for all cell lines; **represents p-values < 0.001. ( D ) The cell surface proteins were labeled with membrane impermeable Sulfo-NHS-Biotin and immobilized on streptavidin-agarose beads. The beads were washed and subjected to SDS-PAGE. E-cadherin was used as a loading control for cell surface proteins. Full-length blots are presented in Supplementary Figure S4 . ( E ) The levels of cell surface proteins were estimated by densitometric scanning of x-ray films using ImageJ software. Shown are fold changes with respect to untransfected cells. Error bars represent means +- s.e.m. from three i

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Experimental details: Figure 10 Claudin-2 expression is limited to the base colon of crypt in NKCC1 WT/DFX and NKCC1 DFX/DFX mice. Representative IF images of NKCC1 WT/WT , NKCC1 WT/DFX , and NKCC1 DFX/DFX mouse colon sections stained with ( A-C ) claudin-2 and ( D-F ) claudin-1 antibodies (red). Sections also show DAPI (blue) or nuclei staining. ( D-F ) In contrast, claudin-1 expression at the lateral membrane was similar in all 3 genotypes. Scale bars : 20 mum. Number of claudin-2-positive cells per crypt were counted on 5 fields, 11 crypts per genotype. There was a significant decrease in the number of cells expressing claudin-2 in the mutant mice. P = .043 for NKCC1-DFX and P = .019 for KO. There was no difference between NKCC1-DFX and NKCC1 KO mice ( P = .93). ( G ) Data are shown as whisker boxes and statistical significance was calculated by 1-way analysis of variance followed by Tukey posttests. CLDN2, claudin-2.

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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.

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Experimental details: FIGURE 9: Cldn2 relocalizes to cell contacts after calcium switch assay more slowly than does cldn1 or cldn4. (A) MDCK II cells cultured on semipermeable membranes were incubated in low-calcium media for 15 h; calcium was added back, and cells were collected and fixed for immunofluorescence at various times after readdition. TER was measured in parallel. (A) Immunofluorescence analysis--(left panel) cldn2, cldn1, and ZO-1 and (right panel) cldn4, ocln, and ZO-1 in untreated monolayers (top images), after 15 h in low calcium (second row), 6 h after calcium readdition (third row), and 24 h after calcium readdition--reveals that by 6 h of calcium return, most the cldn1, cldn4, ocln, and ZO-1 but not cldn2 has returned to cell contacts; the difference in cell-contact localization between cldn2 and 4 is quantified in B. (C) TER measurements show that measurable TER is lost in low calcium but overshoots control levels by more than 10-fold before returning to control values at 24 h.

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Experimental details: Figure 6 Representative confocal images of TJ-related proteins. Cryosections of the jejunal tissue were immunolabeled for ZO-1 and claudin-1, -2, -3, -5, and -7 (in green). Nuclei were visualized with DAPI (in blue). Size bars, 50 mum (small) and 20 mum (large). Abx, antibiotics; CB, C. butyricum ; LR, L. reuteri .

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Experimental details: Fig. 6 GCE significantly disturbs claudin-1 expression in a mouse model. Claudin-1 expression is significantly restored by PAR2-ant and NAC treatment in the lung, as indicated by immunohistochemistry (200 x magnification) (A) and intensity quantification (B). GCE, German cockroach extract; PAR2-ant, protease-activated receptor 2-antagonist; NAC, N-acetylcysteine. * P < 0.01; + P < 0.001. The experiments were repeated 5 times.

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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 experiments (Student'

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Experimental details: Figure 5 Immunofluorescence staining of tight junction (ZO-1, Occludin and Claudin-1) in colon. White arrows point out the tight junction disruption (600 times of magnification). C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate.

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Experimental details: FIGURE 2 Internalization of GFP S. aureus by Normal Human Epidermal Keratinocytes (NHEK). (A) Representative Amnis (r) Image Stream Analysis of NHEK co-cultured with 10 7 CFU/mL FITC LiSA for 1 h at 37degC, then treated for 30 min with 2% penicillin/streptomycin (P/S) to eliminate extracellular bacteria and cultured for a further 4 h prior to staining and analysis. Red stain: Alexa Fluor (r) 647 mouse anti-human cytokeratin 14/15/16/19. Green stain: FITC S. aureus. (B) Representative confocal microscopy 2D image of NHEK after co-culture with 10 7 CFU/mL GFP S. aureus for 1 h at 37degC. (C) Representative immunofluorescent staining of healthy human skin explant after 3 h infection with GFP S. aureus (10 7 CFU/mL) at 37degC. Red: anti-rabbit claudin-1 antibody. Green: GFP S. aureus. Yellow box (left panel) highlights area of right panel magnified. All data are representative of three independent experiments. Images taken at 40x magnification. Scale bars represent 100 mum.

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Experimental details: Figure 5 JMJD1C regulates AMPK in a CAMKK2-dependent manner. (A,B) JMJD1C does not affect activation of LKB1 or levels of its activation partners STRAD and MO25 in NRCMs. The cells were infected with adenovirus expressing Jmjd1c , sh Jmjd1c , or the control constructs for 24 h, followed by Ang II treatment for an additional 24 h. n = 3 in each group. (C) Effects of JMJD1C on CAMKK2 expression. NRCMs were treated as in (A) . ** p < 0.01 vs. Ad-Ctrl or shCtrl. n = 3 in each group. (D,E) Effects of JMJD1C on CAMKK2 mRNA expression. NRCMs were treated as in (A) . ** p < 0.01 vs. Ad-Ctrl or shCtrl. (F) Chromatin immunoprecipitation (ChIP) assay showing the binding of JMJD1C, JMJD2A, and JMJD3 on the FHL1, beta-MHC, and CAMKK2 promoters. IgG was used for negative control, and the enrichment was normalized to the input. *** p < 0.001 vs. IgG. (G) ChIP assay showing that JMJD1C overexpression reduced H3K9me1 enrichment at the CAMKK2 promoter in cardiomyocytes. *** p < 0.001 (H) . The inhibition of CAMKK2 blocks the effects of JMJD1C on AMPK activation in NRCMs. The cells were treated with adenovirus expressing sh Jmjd1c or shCtrl; then the cells were treated with Ang II and the CAMKK2 inhibitor STO-609 (20 muM) for an additional 24 h. All results are from three independent experiments. ** p < 0.01 vs. Ad-shCtrl+PBS; ## p < 0.01 vs. Ad-shCtrl+STO-609. n = 3 in each group.

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Experimental details: Figure 4. Effects of LMWF on the distribution of claudin-1 in the seminiferous epithelium. ( A ) Non-specific staining was detected only in the basal and adluminal compartments of seminiferous epithelium of control sections - negative control. ( B - C ) Immunohistochemical staining of RT transverse cross-sections treated with 0.91% w/v NaCl. ( D - F ) Immunostaining of claudin-1 following treatment with LMWF demonstrated no difference in the distribution of claudin-1 when compared to the control group treated with 0.91% w/v NaCl. Hematoxylin was used for counterstaining.