71-2700

antibody from Invitrogen Antibodies

Targeting: CTNNB1 armadillo, beta-catenin, CTNNB

Provider product page for 71-2700

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [90]
Comments [0]
Validations
Immunocytochemistry [5]
Immunohistochemistry [3]
Other assay [71]

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Product number: 71-2700 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta Catenin Polyclonal Antibody (CAT-15)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody can be used to specifically immunoprecipitate the ~ 92 kDa beta-catenin protein from native cell lysates. It is suitable for use in immunoprecipitation (IP) and IP/western applications. Note that when the antibody is used for straight western blotting, cross-reactivity with a ~100 kDa protein of unknown identity is sometimes observed. Suggested positive control lysates include HeLa and A431.
Reactivity: Human, Mouse, Rat, Chicken/Avian, Xenopus
Host: Rabbit
Isotype: IgG
Antibody clone number: CAT-15
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

CTNNB1 protein structure - 71-2700 shown in red.

Supportive validation

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Experimental details: Human Umbilical Vein Endothelial Cells (HUVEC; C0035C) were grown to confluency in Medium 200 (Product # M-200-500) plus Large Vessel Endothelial Supplement (LVES; Product # A1460801). Cells were fixed with 4% formaldehyde and permeablized with 0.25% Triton X-100 followed by staining with mouse anti-ATP synthase subunit IF-1 monoclonal antibody (Product # A-21355) and rabbit anti-ß-Catenin polyclonal antibody (Product # 71-2700). Primary antibodies were detected with Qdot® 605 Goat F (ab')2 anti-mouse IgG Conjugate (Red; Product # Q11002MP) and Qdot® 655 Goat F (ab')2 anti-rabbit IgG Conjugate (Magenta, Product # Q-11421MP), respectively. Cells were counterstained with NucBlue® Fixed Cell (Blue; Product # R37606) and ActinGreen 488 (Green, Product # R37110) ReadyProbes Reagents. Cover slips were air dried, mounted with Cytoseal 60, and imaged on a Zeiss LSM 710 confocal microscope.

Supportive validation

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Experimental details: HeLa cells were plated on coverslips overnight. The next day the cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 3 µg/mL anti-B-catenin antibody (Product # 71-2700) for 60 min at room temperature followed by three washes with dPBS. Cells were then incubated with a 1:1000 dilution of goat anti-rabbit Qdot® 655 secondary antibody (Product # Q-11421MP) for 60 min, followed by three washes with dPBS. Cells were labeled with NucBlue® Live cell stain (Product # R37605) and ActinGreen™ 488 ReadyProbes® reagent (Product # R37110) according to protocol. Coverslips were then mounted using ProLong® Gold Antifade Reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

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Experimental details: HeLa cells were plated on coverslips overnight. The next day the cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 3 µg/mL anti-B-catenin antibody (Product # 71-2700) for 60 min at room temperature followed by three washes with dPBS. Cells were then incubated with a 1:1000 dilution of goat anti-rabbit Qdot® 655 secondary antibody (Product # Q-11421MP) for 60 min, followed by three washes with dPBS. Cells were labeled with NucBlue® Live cell stain (Product # R37605) and ActinGreen™ 488 ReadyProbes® reagent (Product # R37110) according to protocol. Coverslips were then mounted using ProLong® Gold Antifade Reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HeLa cells were plated on coverslips overnight. The next day the cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 3 µg/mL anti-B-catenin antibody (Product # 71-2700) for 60 min at room temperature followed by three washes with dPBS. Cells were then incubated with a 1:1000 dilution of goat anti-rabbit Qdot® 655 secondary antibody (Product # Q-11421MP) for 60 min, followed by three washes with dPBS. Cells were labeled with NucBlue® Live cell stain (Product # R37605) and ActinGreen™ 488 ReadyProbes® reagent (Product # R37110) according to protocol. Coverslips were then mounted using ProLong® Gold Antifade Reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were plated on coverslips overnight. The next day the cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 3 µg/mL anti-B-catenin antibody (Product # 71-2700) for 60 min at room temperature followed by three washes with dPBS. Cells were then incubated with a 1:1000 dilution of goat anti-rabbit Qdot® 655 secondary antibody (Product # Q-11421MP) for 60 min, followed by three washes with dPBS. Cells were labeled with NucBlue® Live cell stain (Product # R37605) and ActinGreen™ 488 ReadyProbes® reagent (Product # R37110) according to protocol. Coverslips were then mounted using ProLong® Gold Antifade Reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

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Experimental details: Immunohistochemistry analysis of Beta-Catenin showing staining in the membrane of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Beta-Catenin Rabbit Polyclonal Antibody (Product # 71-2700) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

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Experimental details: Immunohistochemistry analysis of Beta-Catenin showing staining in the membrane and cytoplasm of paraffin-embedded human lung adenocarcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Beta-Catenin Rabbit Polyclonal Antibody (Product # 71-2700) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

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Experimental details: Immunohistochemistry analysis of Beta-Catenin showing membrane and weak cytoplasm staining of paraffin-embedded mouse colon tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Beta-Catenin Rabbit Polyclonal Antibody (Product # 71-2700) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

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Experimental details: Immunoprecipitation and Western Blot Analysis: Immunoprecipitation followed by western blot analysis of HeLa cell lysates using rabbit anti-beta-catenin polyclonal antibody (Product # 71-2700).

Supportive validation

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Supportive validation

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Experimental details: FIGURE 2: Msi-1 knockdown by RNAi in Sertoli cells perturbs Sertoli cell-cell junctions via changes in BTB-associated proteins. (A) On day 3, Sertoli cells were transfected with Msi-1-specific siRNA duplexes and nontargeting control siRNA duplexes. The TER was detected until day 7, and each data point is the mean +- SD of n = 5 replicates from a representative experiment. *, p < 0.05. (B) Immunoblot showing the steady-state levels of Msi-1, basal ectoplasmic specialization (ES) proteins, and TJ proteins in lysates of Sertoli cells lysed 48 h after transfection; beta-tubulin served as a loading control. (C) Histogram summarizing selected immunoblotting results in B and normalized against beta-tubulin. Each bar represents the mean +- SD of n = 3 experiments. *, p < 0.05. (D-F) Changes in the localization of TJ proteins, basal ES proteins, and the framework protein vimentin at Sertoli cell-cell interfaces after Msi-1 knockdown were assessed 48 h after transfection. Sertoli cell nuclei were stained with DAPI. Note that considerably less beta-catenin and claudin-11 were detected, and the distributions of occludin and E-cadherin (white arrows) were changed at Sertoli cell-cell interfaces after Msi-1 knockdown. Scale bars: 20 mum.

Supportive validation

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Experimental details: FIGURE 3: An in vivo study assessing the role of Msi-1 in BTB function and spermatogenesis by RNAi intratesticular injection. Msi-1-targeting siRNA duplexes and nontargeting duplexes were administered to each testis in adult mice as a single treatment. The knockdown efficiency, BTB-associated protein expression, biotin tracer experiment, and hematoxylin-eosin (HE) staining at 3, 5, 9, and 12 d postintratesticular injection, respectively. (A) Considerably more intense Msi-1 staining was observed in the Sertoli cells from testes transfected with nontargeting control (a) versus testes transfected with Msi-1-specific siRNA duplexes (b). Scale bars: 20 mum. (B) Histogram summarizing immunoblotting results of Msi-1 and normalized against beta-tubulin. Each bar represents the mean +- SD of n = 3 experiments. *, p < 0.05. (C) Paraffin sections were used to study changes in the expression of beta-catenin (a and b) and claudin-11 (c and d) after Msi-1 knockdown in vivo. The boxed area was randomly selected. Scale bars: 20 mum. (D) The protein levels of beta-catenin and claudin-11 in nontargeting control and Msi-1 knockdown testes. beta-tubulin served as a loading control. (E) Histogram summarizing selected immunoblotting results in D and normalized against beta-tubulin. Each bar represents the mean +- SD of n = 3 experiments. *, p < 0.05. (F) We injected a biotin tracer into the testes of live anesthetized mice ( n = 3) and examined the subsequent changes in BTB integrity after siRNA d

Supportive validation

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Experimental details: Figure 6 Combined effect of Tcf1 and Tcf3-beta-catenin for the Wnt3a response of ES cells (a) Alkaline phosphatase staining of colonies from ES cell lines with different Tcf3 genotypes (indicated on left) and expressing an shRNA (indicated on bottom). Colonies were formed for four days in serum containing media without LIF, and with control or Wnt3a (indicated on top). Percentage of AP+ colonies (white number) represents mean +- standard deviation of biological duplicates. Wnt3a stimulated self-renewal at different levels in Tcf3+/+ , Tcf3-/- and Tcf3DeltaN/DeltaN ES cells, while knockdown of Tcf1 significantly reduced stimulations in all three ES cell lines. (b) Gene expression analysis in different Tcf3 ES cell lines +- Tcf1 shRNA and +- recombinant Wnt3a. The relative level of mRNA for each gene is compared to untreated Tcf3+/+ cells set at 1.0. Numerical values +/- standard deviations are shown in Supplementary Table S5 . (c-e) Chromatin immunoprecipitated with antibodies against Tcf3 (c), Oct4 (d), or beta-catenin (e) was measured by qPCR using primers ( Supplementary Table S5 ) specific for upstream regions of the ten genes labeled at the bottom of graph shown in (e). A representative graph from three biological replicates is shown for each experiment. (c,d) Assays were performed using chromatin from Tcf3+/+ (black) and Tcf3-/- (gray) ES cells. Values represent the mean percentage of DNA immunoprecipitated relative to input +- standard deviations of replicate measuremen

Supportive validation

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Experimental details: Figure 3 CuB inhibits the expression and nuclear translocation of beta-catenin in NSCLC cells. A549 (Panel A ) and H1299 (Panel B ) cells were seeded on glass cover slips for 24 h and then treated with varying concentrations of CuB for 24 h. Endogenous cytoplasmic and nuclear beta-catenin (FITC-green) localization was visualized by immunofluorescence followed by confocal imaging. DAPI was used as nuclear stain (blue). IgG control was used as the negative control. Images are representative of three-independent experiments. The graphs (Panels C , D ) represent the raw integrated density as well as CTCF values in A549 and H1299 cells, respectively. The numbers in parenthesis are indicative of number of cells analyzed for each group.

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Experimental details: Figure 7 beta-catenin is colocalized with both N-cadherin and E-cadherin. Samples were processed for the dual labeling of beta-catenin and either N- or E-cadherin as described in the methods and viewed using a 40x objective. Panels A and B show the dual labeling of beta-catenin (A) and N-cadherin (B) in a field from the outer cortex whereas panels C and D show the dual labeling of beta-catenin (C) and E-cadherin (D) in a similar field. Panels E-F show the same molecules in fields from the inner cortex. E and F: dual labeling of beta-catenin (E) and N-cadherin (F); G and H dual labeling of beta-catenin (G) and E-cadherin (H). Original magnification = 174x.

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Experimental details: Figure 8 Dual labeling of N-cadherin and beta-catenin in the proximal tubule. Panels A and B show that dual labeling of N-cadherin (A) and beta-catenin (B) in the S1/S2 segment of the proximal tubule, whereas panels C and D show the dual labeling of N-cadherin (C) and beta-catenin (D) in the S3 segment of the proximal tubule. Original magnification = 435x.

Supportive validation

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Experimental details: Figure 5 Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier. (A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ~50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ~50-60% knockdown of Rai14 in several pilot experiments, unlike other target genes [ e.g ., Scribble, beta1-integrin, and P-glycoprotein] wherein we could silence the target gene

Supportive validation

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Experimental details: Figure 1 Rai14 is an actin-binding protein in the rat testis. ( A ) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). ( B ) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. ( C ) The specificity of the anti-Rai14 antibody ( Table 1 ) was assessed by immunoblotting using lysates of GC (20 ug protein). ( D ) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins ( e.g ., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48] . ( E ) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-acti

Supportive validation

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Experimental details: Figure 4 CuB inhibits the Wnt/beta-catenin signaling in the NSCLC cells. A549 (Panel A ) and H1299 cells (Panel B ) were treated with 0-100 nM of CuB for 24 h and whole cell lysates were prepared. Western blot analysis was performed to analyze the expression of Wnt/beta-catenin pathway proteins. Blots are representative of three independent experiments. Graphs in (Panel C , D ) represent relative band intensities of proteins in A549 and H1299 cells normalized to beta-actin. The results were obtained from three independent experiments, mean +- SEM. *p < 0.05 versus Control.

Supportive validation

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Experimental details: Figure 5 CuB treatment abrogates TGbeta-1-induced EMT in A549 cells. ( A ) A549 cells were treated with TGF-beta1 (5 ng/mL) for 24 h. TGF-beta1 treated and untreated A549 cells were treated with 25 and 50 nM concentrations of CuB for 24 h. Morphological changes are shown in light microscopic images (100x magnification). ( B , C ) Wounds were created in the sub-confluent cultures of A549 cells and then cells were treated with TGF-beta1 (5 ng/mL) and 25 and 50 nM CuB. Photomicrographs were captured at 0, 12 and 24 h after treatment and wound area was measured. Data were expressed as percent wound closure in the treatment groups compared to control. The results were obtained from three independent experiments, mean +- SEM. **p < 0.01 and ***p < 0.001 compared to untreated control, ## p < 0.01 and ### p < 0.001 compared to respective TGF-beta1-treated control group. ( D ) A549 cells were treated as in (Panel A ), and whole cell lysates were prepared. Western blot analysis was performed to analyze the expression of EMT markers. Blots are representative of three independent experiments.

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Experimental details: Figure 6 Wnt/beta-catenin signaling is involved in the inhibition of metastatic progression of NSCLC A549 cells. A549 (Panels A - D ) were subjected to treatment with 40 nM of Wnt3 or Wnt3a siRNA, respectively. Control- and Wnt3/3a -siRNA transfected cells were treated with the indicated concentrations of CuB for 24 h. Cell lysates were prepared and protein expressions were analyzed by western blot. Blots are representative of three independent experiments. Graphs represent the relative band intensities normalized with beta-actin. A549 (Panels E , F ) cells transfected with Wnt3/3a -siRNA were treated with indicated concentrations of CuB for 24 h. Cellular migration was determined and plotted against percent wound closure in control group. Results were obtained from three independent experiments, mean +- SEM, Statistical significance, *p < 0.05, **p < 0.01 and ***p < 0.001 against control siRNA group.

Supportive validation

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Experimental details: Figure 7 Wnt/beta-catenin signaling is involved in the inhibition of metastatic progression of NSCLC H1299 cells. H1299 cells (Panels A - D ) were subjected to treatment with 40 nM of Wnt3 or Wnt3a siRNA, respectively. Control- and Wnt3/3a -siRNA transfected cells were treated with the indicated concentrations of CuB for 24 h. Cell lysates were prepared and protein expressions were analyzed by western blot. Blots are representative of three independent experiments. Graphs represent the relative band intensities normalized with beta-actin. H1299 (Panels E , F ) cells transfected with Wnt3/3a -siRNA were treated with indicated concentrations of CuB for 24 h. Cellular migration was determined and plotted against percent wound closure in control group. Results were obtained from three independent experiments, mean +- SEM, Statistical significance, *p < 0.05, **p < 0.01 and ***p < 0.001 against control siRNA group.

Supportive validation

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Experimental details: Figure 8 CuB inhibits NNK-induced lung tumorigenesis and Wnt/beta-catenin/MMP-2 signaling. ( A ) Representative images of vehicle-treated, NNK-induced CuB-untreated as well as 0.1 mg/Kg and 0.2 mg/Kg b.w. CuB-treated mice lungs. ( B ) Effects of CuB on lung tumor incidence, lung tumor frequency and relative lung wet weights in NNK-treated mice. mean +- SEM. ### p < 0.001 compared to vehicle-treated lungs, ** p < 0.01 and *** p < 0.001 compared to NNK-induced lungs. ( C ) Tissue lysates from each animal group were used to analyze the expression of different Wnt/beta-catenin signaling markers. beta-actin was used as equal loading control. Blots are representative of three independent experiments.

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Experimental details: FIGURE 4 3,4,5-Tri- O -caffeoylquinic acid (TCQA) stimulated beta-catenin and microphthalmia-associated transcription factor (MITF) expression in human epidermal melanocytes (HEMs). (A) Protein expression of MITF and beta-catenin after 0, 4, 8, and 12 h of treatment with 10 muM of TCQA. (B) Gene expression of MITF after 0, 1, 6, and 12 h of treatment with 10 muM of TCQA. (C) Immunocytochemistry of beta-catenin expression after 0, 4, 8, and 12 h of treatment with 10 muM of TCQA. Scale bar = 25 mum; magnification at 20x. (D) Gene expression of CTNNB1 after 0, 1, 6, and 12 h of treatment with 10 muM of TCQA. (E) Gene expression CTNNB1 after treatment with 10 muM of XAV939 and co-treatment of 10 muM of XAV939 and 10 muM of TCQA for 24 h. (F) MITF gene expression after treatment with 10 muM of XAV939 and co-treatment of 10 muM of XAV939 and 10 muM of TCQA for 24 h. (G) Melanin assay after 48 h of treatment with 10 muM of XAV939 and co-treatment of 10 muM of XAV939 and 10 muM of TCQA for 48 h. The band intensities were done by comparing GAPDH using LI-COR system; and the mRNA level was quantified using TaqMan real-time PCR. Results represent the mean +- SD of three independent experiments. *Statistically significant ( P

Supportive validation

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Experimental details: FIGURE 5 3,4,5-Tri- O -caffeoylquinic acid (TCQA) enhanced microphthalmia-associated transcription factor (MITF) and beta-catenin expression in B16F10 murine melanoma cells. (A) Protein expression of MITF after 12 and 24 h of treatment with 0 and 25 muM of TCQA and 200 nM of alpha-MSH. GAPDH 24 h from Figure 3 band is reused in this figure. (B) Gene expression of MITF after 6 and 12 h of treatment with 0 and 25 muM of TCQA and 200 nM of alpha-MSH. (C) Protein expression of beta-catenin expression after 0, 12, and 24 h of treatment with 25 muM of TCQA. (D) Gene expression of Ctnnb1 after 0, 12, and 24 h of treatment with 25 muM of TCQA. The band intensities were done by comparing GAPDH using LI-COR system; and the mRNA level was quantified using TaqMan real-time PCR. Results represent the mean +- SD of three independent experiments. *Statistically significant ( P

Supportive validation

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Experimental details: Figure 3. The Bcat-regulated genomic program. ( A ) Time course heatmap of Bcat-regulated transcripts. Differential expressed genes from a pairwise comparison of control-MO and Bcat-MO embryos ( > 2 FC, FDR < 5%) at each stage identified 2568 Bcat-regulated transcripts; 1321 downregulated and 1247 upregulated in Bcat-MO embryos. Key genes are color coded based on regional expression from ( D ). ( B ) Venn diagram intersecting Bcat-regulated genes with Bcat-bound genes (and associated peaks) from published X. tropicalis gastrula Bcat ChIP-seq data () identified 898 putative direct targets associated with 2616 peaks. Statistically significant intersections based on hypergeometric tests* p

Supportive validation

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Experimental details: Figure 4. Sox17 and Bcat co-occupy enhancers to regulate the endoderm GRN. ( A ) Eighty-four genes (191 Peaks) are cobound and coregulated by Sox17 and Bcat. Venn diagram show that 415 genes are regulated by both Sox17 and Bcat (left), whereas 2411 genes (3956 peaks) are bound by both Sox17 and Bcat. Gene intersections are significant based on hypergeometric tests, *p

Supportive validation

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Experimental details: Figure 1 Ctdnep1 and Eps8L2 are required for nuclear positioning and cell migration in fibroblasts (A) Representative images of wound-edge 3T3 fibroblasts with or without LPA stimulation in control, Ctdnep1, and Eps8L2 siRNAs stained for beta-catenin (green, cell contacts), pericentrin (red, centrosome), and DAPI (blue, nucleus). The dashed white lines mark the wound edge. (B) Average positions of the nucleus (blue) and centrosome (red) relative to the cell centroid in cells treated with control, Nesprin2G, Ctdnep1, and Eps8L2 siRNAs. (C) Percentage of oriented cells in the conditions analyzed in (B). (D) Average positions of the nucleus (blue) and centrosome (red) in cells treated with Ctdnep1 or Eps8L2 siRNAs and microinjected with KDEL-GFP, Ctdnep1-GFP, Ctdnep1_D67E-GFP (no phosphatase activity), GFP, myc-Eps8L2, or myc-Eps8L2_529-715. (E and F) Average wound closure velocity during wound closure migration (E) and instantaneous cell velocity in random migration (F) in cells treated with control, Nesprin2G, Ctdnep1, and Eps8L2 siRNAs. Data are represented as mean +- SEM. The n value means number of analyzed cells (B, D, and F) or number of experiments (C and E) with >50 cells analyzed per experiment. Statistics was performed by unpaired t test: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale bars 10mum. See also Figure S1 .

Supportive validation

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Experimental details: 6 FIGURE CK1 carried in astrocyte derived EV-IL1beta directly binds to GSK3alpha/beta and APC in target neurons. Neurons were treated with astrocyte derived EV-Vector-CR (ADEV-Vector-CR), astrocyte derived EV-Vector-IL1beta (ADEV-Vector-IL1beta), astrocyte derived EV-CK1-CR (ADEV-CK1-CR), and astrocyte derived EV-CK1-IL1beta (ADEV-CK1-IL1beta) for 18 h. (a) GSK3-Myc-CK1, (b) Axin1-Myc-CK1 and (c) APC-Myc-CK1 were immunoprecipitated and the immunoblot probed for Myc tagged CK1. Myc-CK1 was quantified by band density. (d) Neurons were treated with astrocyte derived EV-CR (ADEV-CR) or astrocyte derived EV-IL1beta (ADEV-IL1beta) for 6 and 18 h, Total GSK-3alpha,beta and -GSK3alpha,beta were detected by Western blot and quantified by densitometry. (e) Neurons were treated with astrocyte derived EV-CR or astrocyte derived EV-IL-1beta for 18 h and active beta-catenin expression was measured by Western blot and quantified by densitometry. (f) Neurons were treated with astrocyte derived EV-CR (ADEV-CR), astrocyte derived EV-IL1beta (ADEV-IL-1beta), astrocyte derived EV-siCK1-IL1beta (ADEV-siCK1-IL-1beta), and Wnt agonist (20 muM) for 18 h. The binding of beta-catenin to Hnrnpc promotor sites at -2.7, -0.3 and +0.2 kb were measured by chromatin immunoprecipitation ChIP-qPCR analysis. Data are mean +- SEM of n = 3-8 independent experiments per condition. * P < 0.05, ** P < 0.01, one-way ANOVA with Tukey post hoc comparisons. (g) Neurons were treated with astrocyte derived EV-CR, astrocy

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Experimental details: Qualitative and quantitative analysis of immunohistochemical staining for N-cadherin (A-C, G), beta-catenin (C-D, H) and c-Src (E-F, I). Representative micrographs of control (A, C, E) and flutamide-treated rats (B, D, F) and a histogram of N-cadherin (G), beta-catenin (H) and c-Src (I) staining intensity expressed as relative optical density ( ROD ) of diaminobenzidine brown reaction products. Immunohistochemical localization of N-cadherin (A-B). Typical distribution of N-cadherin is visible in the basal compartment of the seminiferous epithelium at the blood-testis barrier ( BTB ) (arrowheads, see also the higher magnification) and at the Sertoli cell-elongated spermatid interface (arrows) in testes of control rats (A). In tubules of flutamide-treated rats, increased staining intensity at the BTB (arrowheads, see also the higher magnification) is visible. Note a diffusion of N-cadherin from the BTB site (red bracket) (B). Immunohistochemical localization of beta-catenin (C-D). Typical localization of beta-catenin is visible at the BTB site (arrowheads, see also the higher magnification) and at the Sertoli cells-elongated spermatids interface (arrows) in testes of control rats (C). In tubules of flutamide-treated rats a diffusion of beta-catenin from the BTB site is visible (red bracket) (D). Immunohistochemical localization of c-Src (E-F). Typical localization of c-Src is visible in the basal compartment of seminiferous epithelium at the BTB (arrowheads, see also the higher

Supportive validation

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Experimental details: Immunofluorescent localization of N-cadherin and beta-catenin (A-H), and N-cadherin and c-Src (I-P) in testes of control (A-D and I-L) and flutamide-exposed (E-H and M-P) rats. Corresponding images were merged to show areas of colocalization of N-cadherin and beta-catenin (C, G), and N-cadherin and c-Src (K, O). Scale bars represent 15 mum. Rectangles indicate the location of the higher magnification view. In tubules of control rats, typical localizations of N-cadherin (arrows in A and I), beta-catenin (arrow in B) and c-Src (arrow in J) are visible at the BTB . Note also the colocalization of N-cadherin with beta-catenin (arrow in C) and N-cadherin with c-Src (arrow in K) at the BTB . In tubules of flutamide-treated rats, increased N-cadherin signal is observed (arrows in E and M) as well as diffusion of N-cadherin and beta-catenin signals from the BTB site (arrows and brackets in E, M, and F, respectively). Note altered distribution of c-Src (arrow in N). The colocalization of N-cadherin with beta-catenin and N-cadherin with c-Src is absent, see green and red staining (double arrows in G and O, respectively). Cell nuclei were stained with DAPI (blue; D, H, L and P). Dotted-line circles (D, H, L and P) outline the periphery of seminiferous tubules.

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Experimental details: Representative immunoprecipitation results of adherens junction proteins with c-Src in testes of control and flutamide-treated rats. Lysates of testes from control and flutamide-treated rats were used for coimmunoprecipitation with antibodies against beta-catenin and c-Src. Immunocomplexes were subjected to immunoblotting and stained sequentially with anti-N-cadherin or anti-beta-catenin antibodies. Lysates without immunoprecipitation of normal rat testes (T) were used as positive controls, illustrating antibody specificity. A negative control was also included in which the precipitating antibody was substituted by IgG from the host species of the primary antibody. Experiments were repeated in triplicate.

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Experimental details: Figure 3. Effects of TNFalpha on the steady-state levels of integral membrane, scaffolding and regulatory proteins in Sertoli cells in vitro. Sertoli cells were cultured at high density on Matrigel(tm)-coated 12-well dishes as described in Materials and Methods. On day 4 (designated as 0 h in this figure), TNFalpha (25 ng/ml) was added into Sertoli cell cultures, and cells were terminated at specific time points thereafter for lysate preparation. TNFalpha was dissolved in 10 mM NaH 2 PO 4 pH 7.4 at 22degC containing 0.15 M NaCl and 0.1% BSA (wt/vol). The control consisted of culturing Sertoli cells in media containing an equivalent amount of BSA. ( A ) Immunoblots of selected proteins involved in the regulation and in the maintenance of Sertoli cell barrier function. Actin served as an indicator of equal protein loading. ( B ) Histograms summarizing results shown in ( A ) from at least three independent experiments. Histograms are not shown for proteins whose levels did not change significantly. Each data point was normalized against its corresponding actin data point and then against the protein level at 0 h, which was arbitrarily set as 1. Each bar represents the mean +- SD of n = 3-4 experiments. **p < 0.01 (ANOVA followed by Dunnett's post-hoc test). n.d., not determined.

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Experimental details: Figure 1 PFOS perturbs Sertoli cell BTB function through changes in the expression and/or localization of proteins at the immunological barrier. Sertoli cells cultured on Matrigel-coated bicameral units, dishes or coverslips were treated on day 3 with 10, 20 or 50 muM PFOS for 24 hr. Thereafter, cells were washed twice with PBS to remove residual PFOS and terminated for immunoblotting (IB) or IF on day 4. ( A ) Typical results of a TER experiment that monitored the effects of PFOS, at 20 uM and 50 uM vs . vehicle control (DMSO) on the Sertoli cell TJ-permeability barrier. Each data point is a mean +- SD of triplicate units from an experiment, which was repeated 3 times and yielded similar results. ** P < 0.01 by Student's t -test compared between PFOS-treated and control cells. ( B ) Immunoblot analysis to assess the effects of PFOS on the expression of TJ proteins: occludin, CAR and ZO-1; basal ES proteins: N-cadherin and beta-catenin; actin regulatory proteins: Arp3, Eps8, palladin, formin 1 and plastin 3; signaling molecules: activated Akts: p-Akt1-T308, p-Akt1-S473, pAkt2-S474 vs . Akts (including Akt1, 2 and 3), and activated FAK: p-FAK-Tyr407 vs . FAK; MT regulatory proteins: EB1 and detyrosinated alpha-tubulin. Actin and alpha-tubulin served as protein loading controls. PFOS was found to down-regulate p-Akt1-T308, p-Akt1-S473, p-Akt2-S474 and p-FAK-Tyr407. Composite data of immunoblottings are shown in Figure S1A ; and uncropped blots are shown in Figure S4 . ( C ) Imm

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Experimental details: Fig. 8 GFi1b, beta-catenin and LSD1 co-occupy sites at enhancer regions and gene promoters. a Venn diagram showing GFI1B, beta-catenin and LSD1 ChIP-seq promoter binding prior to and after Wnt3A treatment of K562 cells. P indicates statistical significance of overlap between GFI1B and beta-catenin calculated using Fisher's exact test. b Example of target genes Axin2 and Yaf2 bound by GFI1B, beta-catenin and LSD1 and H3K4me1 and H3K9me2 enrichment before and after Wnt3A treatment. Arrowhead indicates the presence of Gfi1b and TCF binding motifs in the Axin 2 promoter. c Motif enrichment analysis under GFI1B peaks alone (top) and Gfi1b/beta-catenin overlapped peaks (bottom). d GFI1B (top) and beta-catenin (bottom) ChIP-PCR analysis in K562 stable clones expressing GFI1B (S23) or scramble (C5) shRNA (error bars show s.d, n = 3 technical replicates). e Distribution of beta-catenin peaks based on their distance to GFI1B peaks (left panel) and LSD1 peaks (right panel). f Distribution of LSD1 peaks based on their distance to GFI1B peaks (left panel) and reversely (right panel). g Venn diagram showing GFI1B, beta-catenin and LSD1 binding at enhancer regions prior to and after Wnt3A treatment of K562 cells. h Distribution of beta-catenin peaks (left) and their overlaps with GFI1B and/or LSD1 peaks based on the genomic feature of each bound region

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Experimental details: Figure 1 Shear stress (SS) promotes brain microvascular endothelial properties. Confluent monolayers of the brain endothelioma cell line b.End5 were exposed to physiological laminar non-pulsatile SS for 25 and 48 h. SS effects were evaluated by immunofluorescence analysis of the tight and adherens junction proteins ( A ) zonula occludens (ZO)-1 and ( B ) beta-catenin, respectively, which showed a delocalisation of the proteins towards the cell membrane and a decrease in membrane gaps (white arrows). Hoechst 33342 was used as counterstaining for nuclei (blue). Scale bar: 20 um. Semi-quantitative analysis of ZO-1 expression showed ( C ) a slight increase in staining intensity, from 25 to 48 h, and a ( D ) marked decrease in cell membrane gaps, while beta-catenin presented ( E ) a notorious increase in fluorescence intensity, and ( F ) a clear cellular elongation. Data are given as means +- SEM ( n = 3, 10 fields/condition). A Student's t -test for mean intensity and a Mann-Whitney test for membrane gaps and cell elongation were used to evaluate the significant differences, where * p < 0.05 and *** p < 0.001 or $$$ p < 0.001 denote differences between the indicated timepoints.

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Experimental details: Figure 2 Adherens junctions are compromised during interaction between breast cancer cells and brain microvascular endothelial cells. Confluent monolayers of the brain endothelioma cell line b.End5 under physiological laminar non-pulsatile shear stress were exposed to 4T1 breast cancer cells (previously labelled with CellTracker(tm) DMTPX Red Dye) for 1, 3, 6 and 24 h and the expression of the adherens junction protein, beta-catenin, in single and mixed cultures was evaluated by immunofluorescence analysis. ( A ) Analysis of the expression of beta-catenin (green) revealed that the protein is present in both cell types, with a different cellular distribution in mixed cultures as compared with single ones, and further revealed a loss of elongation in b.End5 cells exposed to 4T1 cells (white arrows). Hoechst 33342 was used as counterstaining for nuclei (blue). Scale bar: 20 um. Semi-quantitative analysis revealed ( B ) a decrease in endothelial beta-catenin intensity in mixed culture at 24 h, ( C ) particularly notorious at the cell membrane, which was validated by the ( D ) plot profile analysis of the membrane region pointed out with the yellow brackets. Scale bar: 20 um. The characterisation of 4T1 cells was performed by the quantification of the ( E ) area of tumoural clusters, which increased over time in single cultures, and ( F ) number of clusters, which decreased at 24 h in single culture. Data are given as means +- SEM ( n = 3, 10 fields/condition). A one-way ANOVA was

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Experimental details: Fig. 1 LG3/4/5 protects Sertoli cells from cadmium-induced TJ-barrier disruption. a Preparation of cDNA constructs based on the 80 kDa tail of the laminin-alpha2 chain. The top panel is a schematic drawing that illustrates the functional domains of laminin-alpha2 chain. From the N-terminus, the short arm of laminin-alpha2 is comprised of three globular domains: laminin N-terminal domain (LN), laminin 4a domain (L4a) and laminin 4b domain (L4b). The long arm of laminin-alpha2 is comprised of laminin coiled coil (LCC) domain and five C-terminal laminin-type globular (LG) domains of LG1, 2, 3, 4, and 5. Proteolytic cleavage is close to the N-terminus of LG3 to generate the LG3/4/5 (i.e., the 80 kDa fragment). Six different cDNA constructs, namely LG3 (underlined in blue), LG4 (underlined in red), LG5 (underlined in green), LG3/4 (boxed in pink), LG4/5 (boxed in purple), and LG3/4/5 (boxed in black), were obtained by PCR using Sertoli cell cDNAs as the template and the corresponding specific primer pairs (Table 1 ), with the exception of the LG3/4/5 clone which was obtained by first denaturing LG3/4 and LG4/5, so that these two clones were annealed and then served as the template for PCR by using primer pairs (1) and (6). These constructs were cloned into the mammalian expression vector pCI-neo (Promega). Amino acid residues in orange represent sequences between the different LG domains and the italicized orange residues were not cloned into any of the cDNA constructs, such as LG

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Experimental details: Figure 5 Sevo blocks the PTEN/Akt/GSK-3beta/beta-catenin signaling pathway by modulating the expression of miR-25-3p in HCC cells. HCCLM3 and Huh7 cells were transfected with miR-25-3p mimics or miR-25-3p inhibitor for 24 h followed by Sevo treatment for 6 h. (A) Protein levels of p-Akt, Akt, p-GSK-3beta, GSK-3beta, beta-catenin, c-Myc and MMP9 were measured using western blotting. beta-actin served as the loading control. (B) The bands were semi quantitatively analyzed using ImageJ and normalized to beta-actin density. Data are presented as the mean +- standard deviation. (n=3) of three representative experiments. * P

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Experimental details: Figure 1 Occludin, ZO-1, Cx43, N- and E-cadherin, and beta-catenin mRNA (A to E) and protein (A' to E') expression in yellow semen syndrome (YSS) and white normal semen (WNS) testes, epididymides, and ductuli deferentes. Representative quantitative real-time PCR analysis of occludin (A), ZO-1 (B), Cx43 (C), N- and E-cadherin (D), and beta-catenin (E) mRNA expression in YSS and WNS testes, epididymides, and ductuli deferentes. As an internal control, beta-actin mRNA level was measured. Relative quantification (RQ) is expressed as mean +- standard deviation (SD). Data were obtained from 3 separate experiments. Asterisks indicate statistically significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001; Mann-Whitney U-test). YSS turkeys (n = 6) and WNS turkeys (n = 6). Representative western blots and relative expression of occludin (A'), ZO-1 (B'), Cx43 (C'), N- and E-cadherin (D'), and beta-catenin (E') proteins in YSS and WNS testes, epididymides, and ductuli deferentes. Densitometric analysis of protein content was normalized against the corresponding beta-actin. Protein levels within the WNS tissues were arbitrary set as 1. Data obtained from 3 separate analyses are expressed as mean +- SD. Asterisks indicate statistically significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001; Mann-Whitney U-test). N = 5 each for YSS and WNS tissues.

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Experimental details: Figure 3 Qualitative (A and B) and quantitative (C and D) analysis of immunohistochemical staining for N- and E-cadherin (A) and beta-catenin (B). Representative micrographs of yellow semen syndrome (YSS) and white normal semen (WNS) testes, epididymides, and ductuli deferentes. Counterstaining was performed with Mayer's haematoxylin. Scale bars are 10 mum. Frames indicate the location of the higher magnification view. (A and B) Similar staining pattern for N- and E-cadherin (A) and beta-catenin (B) were observed in the basal compartment at the blood-testis barrier (BTB) (arrows) and in the adluminal compartment of seminiferous epithelium (open arrows) as well as in the lateral plasma membranes of adjacent epididymal principal cells (arrows) and between principal and basal cells (open arrows), and in the lateral plasma membranes of adjacent ductal columnar cells (arrows) and between columnar and basal cells (open arrows) of YSS and WNS epididymides and ductuli deferentes. Note increased N- and E-cadherin and reduced beta-catenin staining intensity at the BTB of YSS testis (see higher magnification views in A and B) and of YSS epididymis and ductus deferens sections (see higher magnification views in B). Note weaker staining for beta-catenin between epithelial principal cells (arrows) and principal and basal cells (open arrows) along the entire YSS epididymis length (see higher magnification inserts in B). Note also sloughed germ cells admixed with mature spermatozoa in YSS ep

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Experimental details: FIGURE 6 Opp and Ppp promote BTB recovery after busulfan treatment in Sertoli cells. (A) Immunofluorescence staining of control, Opp-, and Ppp-treated testes with anti-WT1 antibody (Sertoli cell, green), anti-DDX4 antibody (germ cell, red) and DAPI (blue). White dashed line depicts the basement membrane. (B) Immunofluorescence staining of control, Opp-, and Ppp-treated testes with anti-occludin antibody (BTB, green) and DAPI (blue). Yellow scale markers signify diffusively-distributed occludin signals. White scale markers depict tightly-distributed occludin signals around Sertoli and germ cells. (C) Immunofluorescence staining of control, Opp-, and Ppp-treated testes with anti-beta-catenin antibody (BTB, green) and DAPI (blue). Yellow arrow heads signify diffusively-distributed beta-catenin signals; white arrow heads signify tightly-distributed beta-catenin signals around Sertoli and germ cells. (D) Immunofluorescence staining of control, Opp-, and Ppp-treated testes with anti-Cx43 antibody (BTB, green) and DAPI (blue). Yellow scale markers depict diffusively distributed Cx43 signals; white scale markers depict tightly-distributed Cx43 signals around Sertoli and germ cells. (E) Immunofluorescence staining of control, Opp-, and Ppp-treated testes with anti-ZO-1 antibody (BTB, red) and DAPI (blue). Yellow arrow heads label diffusively-distributed ZO-1 signals. White arrow heads label tightly-distributed ZO-1 signals.

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Experimental details: The integrity of BTB is disrupted in Amh-pa1 -/- mice. a The integrity of the BTB was observed to be destroyed in 8-week-old Amh-pa1 -/- testes. Immunofluorescence using FITC-labeled streptavidin (green) was conducted on seminiferous tubules of Pa1 F/F (upper) and Amh-pa1 -/- testes (lower) which had been injected with biotin beneath the albuginea. Nuclei were stained with DAPI (blue). Dashed boxes showed localization of the enlarged images. Asterisks indicated biotin penetrated seminiferous tubules. b Transmission electron microscope images of adult Pa1 F/F and Amh-pa1 -/- mice testes showing the cleft junctions between Sertoli cells at the basal compartment of seminiferous tubules. Red dashed boxes showed localization of the enlarged images shown in the bottom row. Red dashed lines in the enlarged images indicate the cell-cell junctions. c BTB components are shown to be disorganized in 8-week-old Amh-pa1 -/- mice testis. Immunofluorescence analysis using anti-Claudin11, anti-Occludin, anti-ZO-1, and anti-beta-catenin was conducted in the testis of Pa1 F/F mice (left) and Amh-pa1 -/- mice (right). Nuclei were stained with DAPI (blue). Arrowheads indicate the aberrant patches or diminished signals of the BTB components. Dashed curve indicated basal membrane of seminiferous tubules. d Immunoblot analysis of BTB components, including ZO-1, Occludin, Claudin11, and beta-Catenin, in the testes of adult wild-type mice and Amh-pa1 -/- mice. e Cell junction cannot be formed between

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Experimental details: Nuclear positioning is associated with ER morphology remodeling and requires Climp-63. a FIB-SEM representative image of non-LPA stimulated, non-polarized wound-edge U2OS cell. One stack of 998 SEM images from one leading edge cell. b Representative images of perinuclear (yellow insets) and peripheral (green insets) regions. The ER is highlighted in red, light red represents tubules (1 um). Measurements represent the depth of the section. c as in a , but an LPA stimulated and polarized cell. One stack of 1798 SEM images from one leading edge cell. d as in b , but an LPA stimulated and polarized cell. e Representative widefield epifluorescence images of wound-edge NIH3T3 fibroblasts. Cells were immunostained for cell-cell contacts (purple; beta-catenin), centrosome (white; pericentrin) and DNA (DAPI, blue). f Nuclear positions relative to the cell centroid of cells represented in e . Significance (Two-tailed unpaired t-test) was calculated between experimental condition and the scramble siRNA with LPA. **** p < 0.0001 (Scramble no LPA, Nesprin-2G, Climp-63 #1, Climp-63 #2, Triple KD); * p < 0.05. Box shows first quartile, median and third quartile, and whiskers show 10-90% percentile. Experiments were repeated >=3, except ( a - d ). Scale bars: a , b , c , d 1 um; e 40 um.

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Experimental details: Figure 9 HBMEC treatment with selected molecules proved lack of cytotoxicity for 3 candidates. HBMEC were exposed to each of the selected molecules (8, 17, 19, 20, 25, and 27), or no addition (control; Ctr), at a concentration of 100 uM, for 24 h, for viability assays ( A ), or at U87MG EC 50 , during 9 h, for BBB integrity ( B ) or cytoskeleton ( C ) analyses. ( A ) Cell viability was assessed by MTT assay and the values are presented as a percentage relative to control. Molecules with significant cytotoxic effects are presented as pattern-filled bars, and innocuous molecules are shown as white bars. ( B ) BBB integrity was assessed by immunostaining of beta-catenin, with nuclear counterstaining by Hoechst 33342. ( B1 ) Arrows point to gaps in the HBMEC monolayer caused by incubation with toxic molecules. ( B2 ) Semi-quantitative analysis of endothelial gaps, where values are presented as percentage of control. ( B3 ) 3D representative plots of beta-catenin expression obtained by counting the number of image pixels per area in disrupted monolayers. ( B4 ) Semi-quantitative analysis of beta-catenin internalization by fold-change of mean image pixels (Px) in the membrane and interior of treated cells vs. control. ( B5 ) Morphological analysis of HBMEC roundness by cells delineation. ( C ) Cytoskeleton analysis was performed based on F-actin labeling. ( C1 ) Arrows highlight the presence of stress fibers. ( C2 ) Cytoskeleton stress fibers profile per cell representation by semi

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Experimental details: Figure 3 KW-2478 and Mol6 enhance barrier properties through increased membrane beta-catenin expression in b.End5 cells exposed to 4T1 cells. Confluent monolayers of b.End5 cells under physiological laminar non-pulsatile shear stress were exposed to 4T1 cells (previously labelled with CellTracker(tm) CMFDA Green Dye,) and treated with KW-2478 or Mol6 (1 uM), or DMEM (untreated), for 24 h and the expression of the AJs protein beta-catenin (red), in single and mixed cultures, was evaluated by immunofluorescence analysis. Hoechst 33342 was used as counterstaining for nuclei (blue). Scale bar: 20 um. ( A ) Treatment with KW-2478 and Mol6 induced an increase in beta-catenin expression in endothelial cells exposed to 4T1 cells, particularly at cell membrane (white arrows), and an increase in 4T1 cells with aberrant nuclei (white arrow heads). Semi-quantitative analysis of endothelial beta-catenin ( B ) mean intensity per cell, ( C ) nuclear intensity, and ( D ) membrane intensity confirmed the observations. ( E )The localisation of beta-catenin was studied through plot profiles of pixel intensity throughout an endothelial cell (edge to edge). Semi-quantitative analysis of tumour cell cultures confirmed ( F ) an increase in the number of aberrant nuclei upon treatment with each drug, as well as ( G ) a decrease in beta-catenin mean intensity per cell. Data are represented as mean +- SEM of three independent experiments, where 3 cells/field, 5 fields/condition were evaluated. Statist

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Experimental details: Figure 4 BKM120 induces endothelial barrier properties through the increase in membrane beta-catenin expression. Confluent monolayers of b.End5 cells under physiological laminar non-pulsatile shear stress were exposed to 4T1 cells (previously labelled with CellTracker(tm) CMFDA Green Dye) and treated with BKM120 or FTY720-P (0.1 uM), or DMEM (untreated), for 24 h and the expression of the AJs protein, beta-catenin (red), in single and mixed cultures was evaluated by immunofluorescence analysis. Hoechst 33342 was used as counterstaining for nuclei (blue). Scale bar: 20 um. ( A ) Treatment with BKM120 or FTY720-P induced an increase in beta-catenin expression in endothelial cells exposed to 4T1 cells, particularly at cell membrane (white arrows). Semi-quantitative analysis of endothelial beta-catenin ( B ) mean intensity per cell, ( C ) nuclear intensity, and especially ( D ) membrane intensity validated these observations. ( E ) The localisation of beta-catenin was studied through plot profiles of pixel intensity throughout an endothelial cell (edge to edge). Semi-quantitative analysis of tumour cell cultures revealed ( F ) no aberrant nuclei upon treatment, but ( G ) a decrease in beta-catenin mean intensity per cell after FTY720-P treatment. Data are represented as mean +- SEM of three independent experiments, where 3 cells/field, 10 fields/condition were evaluated. Statistical significances are denoted as * p < 0.05 and *** p < 0.001 vs. untreated conditions within the same

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Experimental details: Figure 5 MH induces endothelial barrier properties through the increase in membrane beta-catenin expression. Confluent monolayers of b.End5 cells under physiological laminar non-pulsatile shear stress were exposed to 4T1 cells (previously labelled with CellTracker(tm) CMFDA Green Dye) and treated with MB or MH (1 uM), or DMEM (untreated), for 24 h and the expression of the AJs protein beta-catenin (red) in single and mixed cultures was evaluated by immunofluorescence analysis. Hoechst 33342 was used as counterstaining for nuclei (blue). Scale bar: 20 um. ( A ) Treatment with MB and MH induced an increase in beta-catenin expression in endothelial cells exposed to 4T1 cells, particularly at cell membrane (white arrows), and an increase in 4T1 cells with aberrant nuclei (white arrow heads). Semi-quantitative analysis of endothelial beta-catenin ( B ) mean intensity per cell, ( C ) nuclear intensity, and especially ( D ) membrane intensity confirmed the observations. ( E ) The localisation of beta-catenin was studied through plot profiles of pixel intensity throughout an endothelial cell (edge to edge). Semi-quantitative analysis of tumour cell cultures revealed ( F ) an increase in aberrant nuclei upon treatment with each drug, as well as ( G ) a decrease in beta-catenin intensity per cell. Data are represented as mean +- SEM of three independent experiments, where 3 cells/field, 10 fields/condition were evaluated. Statistical significances are denoted as * p < 0.05 and *** p

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Experimental details: Figure 5. G. intestinalis secreted cysteine proteases have effects on epithelial barrier proteins. A, B, C. Levels of tight junction and adherence junction proteins in Caco-2 whole cell extracts were examined after 24 h of recombinant cysteine proteases treatment (A, 14019; B, 16779; C, 16160), respectively, by Western blot analysis, with GAPDH used as a loading control. Barrier proteins were detected with a mouse or rabbit anti-barrier protein antibody and then incubated with a HR- conjugated mouse or rabbit antibody. D E, F. The localization of occludin in Caco-2 cells visualized by immunofluorescence microscopy. Caco-2 monolayers were incubated with 2.5 ug/ml of 14019 (D), 16779 (E), 16160 (F) in the absence or presence of E64 (10 uM) for 24 h, followed by fixation of PFA. Occludin was detected with a mouse anti-occludin antibody and then incubated with an Alexa Fluor 488-conjugated mouse antibody. Images were taken by a Zeiss Axioplan II Imaging fluorescence microscope and analyzed by ZEN 2.1 software. Bar = 50 um.

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Experimental details: Figure 3C-E TCQA enhanced beta-catenin expression in the hair follicle. ( C ) beta-catenin protein expression was determined at the end of the treatment period. The protein was extracted from the treated area from the mice dorsal part, and western blot was carried away. ( D ) Band intensities was done assessed using LI-COR system. Results represent the mean +- SD of three independent experiments. *Statistically significant ( P

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Experimental details: Figure 3 Aroclor1254 influenced distribution of junction proteins in primary SCs. Primarily isolated SCs were cultured on matrigel-coated coverslips at 0.05 x 10 6 /cm 2 . On day 3, Aroclor1254 (10 mu g/mL), SB203580 (10 mu m ), or the mixture of the above two was added to the medium. After 24 h, the cells were fixed with 4% paraformaldehyde. The distribution of BTB proteins at cell-cell junctions was observed by immunofluorescence using Alexa Fluor 488-conjugated secondary antibody (green) with cell nuclei stained with DAPI (blue). A reduced signal of occludin and disturbed distributions of N-cadherin and beta -catenin were detected at cell junctions of Aroclor1254-treated SCs, as compared with the sharply defined signals in the control and the Aroclor1254+SB203580 groups. Scale bar=25 mu m, which applied to all micrographs in Figure 3

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Experimental details: Figure 5 The effects of Aroclor1254 on the barrier function of SC epithelium after inhibiting the caveolin-dependent endocytosis. The SCs were treated with or without Aroclor1254 in the presence or absence of CO as the regimen depicted in Supplementary Figure S2a . ( a and b ) The barrier function of SC epithelium measured by recording TER ( a ) or calculating the permeability of Na-F ( b ) across the cell monolayer. The Na-F permeability in the vehicle control on day 3 was arbitrarily set at 100%. CO treatment was found to partially resist the disruption of BTB by Aroclor1254. Each bar refers to mean+-S.D. of n =3 experiments using different batches of SCs. ** P

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Experimental details: Fig. 7 Wnt/beta-catenin signaling induces cultured CF activation and ECM gene expression downstream of Tgfbeta signaling in vitro. a Representative images of alphaSMA ( green ) staining in cultured P60 CFs under indicated conditions are shown. Scale bar = 100 mum. b Wnt1 (100 ng/mL) and TGFbeta1 (10 ng/mL) induces significantly more alphaSMA + CFs relative to DMSO controls in cultured P60 CFs. Inhibition of Wnt/beta-catenin signaling by XAV939 (5 muM) abrogated Wnt1-induced alphaSMA expression in CF and also partially suppressed TGFbeta1-induced alphaSMA expression. * P < 0.05 vs. DMSO groups, + P < 0.05 vs. Control. c mRNA expression of the Wnt target gene Axin2 , and ECM genes Col1a1 , Col3a1 , Postn , and Acta2 in P60 CFs with indicated treatments was measured by quantitative PCR. * P < 0.05 vs. control, + P < 0.05 vs. DMSO. N = 4 independent cultures per group. Statistical significance was determined by Kruskal-Wallis tests followed by Mann-Whitney U tests for pairwise comparisons using Bonferonni adjustments to control for multiple testing. d ChIP assays were performed using P0 cultured CFs treated with BIO (2 muM) to determine the binding of beta-catenin in protein complexes with TCF/LEF consensus containing proximal sequences of Axin2 , Col3a1 , and Postn genes. Compared to IgG negative control, binding of beta-catenin to Col3a1 and Postn was significantly enriched. Axin2 was used as positive control. N = 4 independent cultures per group. Fold enrichment of DNA binding