34-9100

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HT 29 (Lane 1), HCT 116 (lane 2), and NIH/3T3 (Lane 3). The blots were probed with Anti-Claudin-7 Rabbit Polyclonal Antibody (Product # 34-9100, 2 µg/mL) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A17 kDa band corresponding to Claudin- 7 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by iBlot® 2 Dry Blotting System (Product # IB21001).The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Western blot was performed using Anti-Claudin 7 Polyclonal Antibody (Product # 34-9100) and a 22 kDa band corresponding to CLDN7 was observed in HT-29, HCT 116, Caco-2 and Mouse Placenta and not in Panc-1 and Mouse Heart which are reported to be less expressing for CLDN7. Membrane enriched extracts (30 µg lysate) of HT-29 (Lane 1), HCT 116 (Lane 2), Caco-2 (Lane 3), Panc-1 (Lane 4) and tissue extracts (30 µg lysate) of Mouse Heart (Lane 5) and Mouse Placenta (Lane 6) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1.5 ug/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

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Experimental details: Figure 2 Loss of HOXA5 disrupts cell-cell adhesion molecules that govern epithelial integrity A . Gene set enrichment analysis of MCF10A-scr and HOXA5 depleted cells indicates that cell-cell junction genes are enriched for expression in MCF10A-scr cells. B . RT-PCR analysis shows reduced expression of EMT markers (CDH2 and CDH3), tight junction molecules (Occludin and CLDN7) and increased expression of luminal fate marker, FOXA1 and reduced expression of FOXQ1 in MCF10A-scr, HOXA5 depleted MCF10A-sh528 and MCF10A-sh529 cells. C . Western blot analysis of HOXA5 and HOXA5-regulated genes CDH1, Occludin, CLDN7 and CLDN1 in MCF10A-scr, -sh528 and -sh529 cells. beta-actin serves as the loading control. D . Western blot analysis shows decrease in CDH1 and CLDN1 protein upon HOXA5 depletion in MCF10A cells; and reversal by ectopic expression of HOXA5 in the knock-down clones. E . Representative immunofluorescence images of CDH1 (Green), Occludin (Yellow), actin stress fiber, Phalloidin (Red) and nuclear marker, DAPI (Blue) with expanded views (2x) of CDH1 and Occludin in MCF10A-scr cells to clearly visualize localization.

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Experimental details: Figure 6 Retinal-induced cell differentiation in MCF10A is compromised when HOXA5 is repressed A . Western blot analysis of HOXA5 and CDH1 expression in MCF10A-tet-sh528 cells with dox-induced depletion of HOXA5 over 96 hours. beta-actin serves as the loading control. B . Flow cytometry analysis of CD24 expression in MCF10A-tet-HOXA5 with doxycycline-induced depletion of HOXA5 shows decrease of CD24+ population (7-AAD negative) from 47% to 24% over 96 hours. C . Flow sorted CD24 - population was treated with DMSO, 1 uM retinal (ATAL) or 1 uM ATAL + 100 nM doxycycline (DOX) and cultured for 1 week. Flow cytometry analysis of CD24 (x-axis) and CD44 (y-axis). Quantitative analysis is shown in the bar graph. D . Quantitative RT-PCR analysis of HOXA5, CD24, CDH1, Occludin, CLDN7 and ALDH1A3 in MCF10A-tet-sh528 cells treated with DMSO, 1 uM retinal (ATAL) or 1 uM ATAL + 100 nM doxycycline (DOX) and cultured for 1 week. E . Western blot analysis of HOXA5, CDH1, p21, ZO-1, Occludin, CLDN7 and CK18 in cells treated with DMSO, ATAL and ATAL+ doxycycline as in (D). beta-actin serves as the loading control.

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Experimental details: Figure 1 Comparison of expression levels of claudins- 1 (1A), 2 (1B), 4 (1C) and 7(1D) in Misoprostol treated (M+) (samples 1+ to 7+) versus untreated (M-) (samples 1- to 5-) cervix . Representative immunoblots in the top panels whilst box plots, lower panel, represent mean (SE) relative optical densities (ROD) for all studied tissues: M+ (n = 10) vs. M- (n = 5). Block arrows showing Gbetaeta as internal loading control at 35 kDa. C-positive control in the first lane. C-1 = claudin-1; C-2 = claudin-2; C-4 = claudin-4; C-7 = claudin-7. ROD- relative optical density in Arbitrary Units (AU).

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Experimental details: Figure 3 Representative confocal photomicrographs comparing localization patterns of claudins-1 (C1), -2(C2), -4(C4) and -7(C7) and occludin (Ocld) in Misoprostol treated (M+, right panel) and untreated (M-, left panel) cervix . The FIT-c (green) and Cyanine-3 (Cy-3-red) labelled secondary antibodies were used; DAPI (blue) for nuclear staining with overlays of red, green and blue (RGB) channels. In the untreated cervix, occludin (red) shifted to intermediate and more superficial layers (3B-red). In the untreated cervix, occludin (3A-red) and claudin-2 (3A green) were expressed in basal layers only (3A-overaly) but following Misoprostol treatment, de novo expression of claudin-2 (3B-green) in the intermediate layer resulted in both now localizing in the basal and intermediate layers (3B-overlay). Compared to diffuse lattice pattern in basal to intermediate layers in the untreated cervix (3C red), Misoprostol induced expression of claudin-7(3D red) in basal and intermediate layers. Claudin-4 was expressed mainly in the nuclei throughout the ectothelium (3C green) in the untreated cervix, with de novo cytoplasmic expression in Misoprostol-treated tissue (3D & 3F green). Claudins 1 and 4 exhibit nuclear and plasma membrane expressions in Misoprostol-treated cohort (3F). Arrows at basement membranes; S-stroma, E-epithelium. 3A- 2 x 2 tile; x 20air objective. NA-0.8, Bar = 8 mum.

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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 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 1 Experimental workflow and characterization of SW480-ADH Mock and Snail1 cells. (A) Scheme of the workflow followed in this study. (B) Representative phase-contrast micrographs (upper panels) and confocal laser immunofluorescence images showing F-actin staining (lower panels) of SW480-ADH Mock and Snail1 cells. Scale bar, 20 um (upper panels) and 10 um (lower panels). (C) Western blot analysis of whole-cell extracts from SW480-ADH Mock and Snail1 cells showing Snail1 overexpression and its effect on the expression of epithelial (E-cadherin, Occludin and Claudin-7) and mesenchymal (Lef-1) markers. beta-Tubulin was used as a loading control.

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Experimental details: Figure 3 Western blot detecting claudin-7 and ss-actin . The upper panel shows claudin-7 in samples from a colorectal cancer patient. Normal distant is a sample taken as far away from the tumour as possible in the surgically removed tissue. Normal adjacent is a sample taken just adjacent to the tumour and CRC is from the cancer itself. The lower panel shows the loading control (ss-actin).

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Experimental details: Figure 4 Immunohistochemical staining for claudin-7 in colorectal adenomas, carcinomas and adjacent normal tissue . The top four pictures are taken from an individual with dysplasia but no record of carcinoma and the bottom four pictures are from a patient with colorectal cancer. A) Tissue section including both normal mucosa and dysplastic tissue, stained with PAS/Alcian. B) Neighbouring tissue section stained for claudin-7. The white arrow indicates mucosa of normal appearance and the black arrow indicates dysplastic tissue. C) High magnification of the normal mucosa from B, showing staining mainly at the basolateral cell membranes of the epithelial cells. D) High magnification of the dysplastic tissue from B showing a patchy staining pattern with areas of low and normal staining. E) Tissue section with cancerous tissue to the right and normal mucosa to the left, stained with Hematoxylin and Eosin. F) Neighbouring tissue section stained for claudin-7. The white arrow indicates the mucosa with normal appearance. The black arrow points out carcinomatous tissue. G) High magnification of histologically normally appearing mucosa from F, showing staining mainly localised to the basolateral cell membranes of the surface epithelial cells. H) High magnification of carcinomatous tissue showing faint, patchy staining of the epithelial strands of tumour tissue. Scale bars: 500 mum (A + B + E + F), 100 mum (C + D + G + H).

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Experimental details: Figure 7 Autocrine CSF1R signaling maintains low expression of tight junction proteins and luminal keratins in claudin-low breast cancer cells A. MDA-MB-231 cells were treated in vitro with either control or the inhibitory antibody to human CSF1R (aH-CSF1R), in the presence of TGFbeta. After 48hours, mRNA expression of a panel of luminal keratins and tight junction proteins was assessed by real-time PCR. Results are shown as average mRNA expression relative to control treatment. N=3 separate experiments with duplicate plates. B. MDA-MB-231 orthotopic tumors were treated in vivo with either control IgG or the inhibitory antibody to human CSF1R (aH-CSF1R). After 48hours, tumor cells were isolated by FACS (due to GFP stable expression in the MDA-MB-231 cells), and total RNA was extracted. mRNA expression of a panel of luminal keratins and tight junction proteins was then assessed by real-time PCR. Results are shown as average mRNA expression relative to control treatment. N=3 tumors per condition. C. Western blot to assess protein levels after MDA-MB-231 cells treatment in vitro as described in panel A. Beta-actin was used as a positive control. Representative images are shown from two separate experiments. D. Immunohistochemistry of tumor sections for keratin 8 (KRT8) and claudin 7 (CLDN7) in the tumors treated as described in panel B. Representative images are shown from three separate tumors per group. E. In vitro invasion was measured in matrigel-coated transwells for contro

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

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

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Experimental details: Figure 3. Expression levels of CLDN7, APLP2 proteins and their localization in OVCA420 cells. (A) Immunoprecipitation of CLDN7 and APLP2. OVCA420 cells were grown for 72 h and cell lysates were immunoprecipitated with anti-CLDN7, anti-APLP2 and anti-GAPDH antibodies. Proteins were visualized by immunoblotting with an anti-APLP2 and anti-CLDN7 antibodies. Total cell lysate serves as a positive control and no primary antibody in immunoprecipitation serves as a negative control. (B) Immunoblotting analysis of CLDN7 and APLP2 after siRNA-mediated knockdown of CLDN7 and APLP2. GAPDH was used as a loading control. (C) Immunofluorescence of OVCA420 cell line shows strong expression of CLDN7 mainly at cell junction and APLP2 expression at cell junctions as well as cytoplasm. CLDN7 and APLP2 also show colocalization at cell junctions. After fixing in methanol, OVCA420 cells were incubated with primary CLDN7 and APLP2 antibodies and visualized with secondary antibodies conjugated to Alexa fluor (Red color represents APLP2 and green color represents CLDN7). Nuclei were counterstained with DAPI.

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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 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: Fig 5 Levels and localization of tight junction protein Claudin-7 are regulated by EpCAM in MDCK cells. (A) EpCAM-depleted MDCK lines Esh1, Esh2 and control shRNA line ctrl sh were SDS extracted one day after plating at subconfluent cell density. Protein levels of Claudin-7 and of GAPDH as a loading control were analyzed in the same immunoblot. The graphs show quantification of Claudin-7 protein normalized to GAPDH levels in the same sample. Error bars: S.E.M. of four samples for each cell line; p values derived from unpaired Student's t test: ** p = 0.0042 for Esh1 to ctrl sh and ** p = 0.0057 for Esh2 to ctrl sh. (B) Claudin-7 protein levels in MDCK cells and cell lines MhE16, MhE33 overexpressing human EpCAM were analyzed as described in (A). Arbitrary units for protein intensities in Y-axis (AU) x10 3 ; error bars: S.E.M. of three samples for each cell line; p value derived from unpaired Student's t test: * p = 0.042 for MhE16 to MDCK. (C) Confocal images were acquired of cells 24 hours after plating on IBIDI chambers and 0 hours after removal of the stencil. The figure shows maximum intensity projections and orthogonal projections of confocal stacks of MDCK, MhE16 and Esh2 cells stained for nuclei (blue), EpCAM (green), and Claudin-7 (red). In the MDCK and MhE16 lines, both EpCAM and Claudin-7 localize along basolateral membrane. In the Esh2 line, Claudin-7 does not distribute throughout the basolateral membrane, and EpCAM staining is very weak. In Esh2 line, greater con

Supportive validation

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Experimental details: Fig 6 Rescue of Claudin-7 protein levels by expression of EpCAM-YFP or point mutants of EpCAM-YFP in MDCK cells depleted of EpCAM. (A) Schematic of EpCAM-YFP (EY) and EpCAM-YFP mutant proteins (EIY and EEY). Extracellular domain (EC), transmembrane domain (TM), intracellular domain (IC) and YFP fusion are indicated. Approximate position of amino acids mutated in EIY and EEY are indicated with their one letter codes. (B) EpCAM-depleted Esh2-derived cell lines expressing EY, EIY or EEY were plated at confluent density onto an IBIDI 4 well dish for one day and YFP fluorescence was imaged before well chambers were removed for a migration assay. YFP fluorescence localizes to cell-cell contacts in all three cell lines. Scale bars: 50 mum. (C) Indicated MDCK cell lines were cultured, extracted and protein levels were analyzed as described for Figs 1A , 2A and 5 . EpCAM-YFP fusion proteins EY, EIY and EEY appear as double bands; MDCK-endogenous EpCAM (ME) is a single band. EEY appears slightly larger and is poorly recognized by the EpCAM extracellular domain antibody (top EpCAM blot). Expression of Claudin-7 is reduced in the parental Esh2 line (Esh) compared to MDCK and control shRNA line ctrl sh (Claudin-7 blot; see also Fig 5 ). (D) Graphs show quantifications of indicated proteins normalized to GAPDH levels in the same sample. For EpCAM protein levels, residual MDCK-endogenous EpCAM (ME) and EpCAM-YFP protein double band levels (EY, EIY, EEY) were combined to show total EpCAM lev

Supportive validation

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Experimental details: 2 FIGURE Dapiglutide strengthens barrier function in the jejunum but not in the colon. (A) FITC-4 kDa dextran permeability of stripped jejunum was measured in the Ussing chamber for n = 6-8 mice, mean +- SD, * p < 0.05. (B) FITC-4 kDa dextran permeability of stripped colon was measured in the Ussing chamber for n = 4-5 mice, mean +- SD. (C) Upper panel: representative western blot of claudin-7 in jejunum tissue. Lower panel: corresponding densitometric analysis of relative claudin-7 protein content, n = 3-4 mice, mean +- SEM, ** p < 0.01. (D) Upper panel: representative western blot of claudin-7 in colon tissue. Lower panel: corresponding densitometric analysis of relative claudin-7 protein content, n = 4-5 mice, mean +- SEM, * p < 0.05. (E) Immunofluorescence images of jejunum sections: claudin-7 in green, F-actin in red, and nuclei in blue. Scale bar: 50 mum. (F) Immunofluorescence images of colon sections: claudin-7 in green, F-actin in red, and nuclei in blue. Scale bar: 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: 2 Western blotJian Ce Fei Yan Zu Zhi Ji Qi Yan Pang Zu Zhi Zhong Claudin-7He SlugDe Biao Da . A:Fei Lin Yan , Xian Yan Zhong Claudin-7He SlugDe Dan Bai Biao Da Qing Kuang ;T1, 2:SCC;T3, 4:ADC;N:Yan Pang Zu Zhi ;B:Claudin-7He SlugDan Bai De Xiang Dui Biao Da Liang . * : P < 0.05. Expression of Claudin-7 and Slug protein in lung can-cer tissues and paracancerous specimens by Western blot. A: Result of Western blot for expressions of Claudin-7 and Slug in lung quamous cell carcinoma and adenocarcinoma; T1, 2: SCC; T3, 4: ADC; N: paracancerous specimens; B: The histogram of relative expression rate of Claudin-7 protein compared to Slug. * : P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Development of intestinal metaplasia and ectopic gastric gland in aged stCldn18-/- mice. ( A ) Expression levels of Cldns in the stomach from s tCldn18+/+ (n = 6), and stCldn18-/- (n = 7), mice at 100 w.o. quantified by qRT-PCR. The expression levels of Cldn2 , 4 , 7 , and luCldn18 were significantly increased in the stomach of stCldn18-/- mice at old age. Gene expressions were normalized to GAPDH. Results are expressed as mean +- SD. Comparisons between 2 groups were performed using Student's t test, and differences with P < .05 were considered statistically significant. n.s., not significant. * P < .05. ( B ) Immunofluorescence micrographs for Cldn2 or Cldn7 ( green ) co-stained with E-cadherin ( red ) and with or without villin ( blue ) in the stomach from stCldn18+/+ and stCldn18-/- mice (around 50 w.o.). Representative images from at least 3 independent experiments are shown. Scale bars = 100 mum. ( C ) Hematoxylin and eosin-stained images of the ectopic gastric gland in aged stCldn18-/- mice. Ectopic gastric glands were found in 1 of 3 old stCldn18-/- mice by examining stomach tissue slices. Right panels are magnified images of the boxed regions in the left panels. Representative images from at least 2 independent experiments are shown. Scale bars = 100, 50, 10 mum, left to right panel. ( D ) ( Left ) Immunofluorescence micrographs for TFF2 ( green ) and Pepsin C ( blue ) as SPEM cell markers co-stained with CD44 (red) and DAPI ( white ) in the ectopic gastric

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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 )