e-cadherin Search Results


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  • 99
    Thermo Fisher n cadherin
    N Cadherin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc e cadherin
    Calcipotriol suppressed the activated HSCs-induced tumor progression of heat-treated residual HCC cells in vivo. a , b The changes of Snail, Vimentin, <t>E-cadherin</t> and N-cadherin, PCNA expression in heat-exposed residual HCC cells (MHCC97H, Hep3B, HepG2 and Huh7) cultured with conditioned medium (CM) from HSCs or calcipotriol-treated HSCs were compared. HSCs cells (LX2 and pHSC) were stimulated by the pre-treatment with TGF-β1. c The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-treated residual HCC cells were reversed when CM from calcipotriol-treated LX2 cells was supplemented with 100 ng/mL POSTN. d Calcipotriol inhibited tumor growth compared with the control group. The combination therapy of calcipotriol and cisplatin significantly reduced tumor growth. e The expression of POSTN, α-SMA, PCNA, E-Cadherin and cleaved-Caspase-3 was detected by immunohistochemical analysis. Calcipotriol, Cal. ** P
    E Cadherin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 15869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam e cadherin
    Effect of Carved on hepatic contents of ( A ) <t>E-Cadherin</t> and ( B ) vimentin. Values are expressed as mean ± S.D (n = 7). Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, P
    E Cadherin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 6901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson e cadherin
    has/apkc regulates polarised localisation of <t>E-cadherin</t> independent of peridermal cell height. Regression analysis of normalised E-cadherin intensity plotted against normalised cell-height ( A ) performed on data presented in Figure 2A4 . The red line marks the regression line and red shaded area marks the 99% prediction limits as per the sibling data. The points outside the red area are considered to have abnormal distribution. The wild type siblings (WT sib) and has/apkc mutant cells are classified according to the height as tall, medium or short cells ( B ). The apical perimeter was reduced in both tall and medium/short cells ( C1, D1 ) in has/apkc mutant embryos. A graphical representation of E-cadherin localisation along the apicobasal axis ( C2, D2 ) and the noise index ( C3, D3 ) in has/apkc mutants show a decrease in robustness of E-cadherin polarity in both tall ( C2, C3 ) and medium/short cells ( D2, D3 ). Asterisk indicates significant difference (p
    E Cadherin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 9399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology e cadherin
    Different kinetics for disruption of desmosomes and adherens junctions by EGF. (a) SCC 12F cells were treated with EGF for the indicated times, fixed, and then probed with <t>E-cadherin</t> or desmoglein-2 antibodies. Note the relocalization of the desmosomal cadherin desmoglein-2 from the cell borders following treatment with EGF for 4–6 h (white arrows). This pattern differs from that observed for the adherens junctional cadherin, E-cadherin, where strong border staining is evident at 6 hours post EGF treatment (white arrowheads). (b) Cells were treated with EGF, fixed, and then stained with phalloidin to stain the actin cytoskeleton, or with a pan-cytokeratin antibody, to label keratin filaments. Disorganization of the keratin network is seen at 6 hours (white arrow), while the actin cytoskeleton remains intact at 8 hours posttreatment (white arrowheads).
    E Cadherin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 7703 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology ve cadherin
    Notch1 is activated in response to shear stress by endocytosis of Dll4 a. ICD cleavage as measured by western blot with an antibody specific to cleaved ICD (N1 V1754) in Scramble and Dll4-KO ECs under flow. b , Fluorescent micrographs of Scramble and Dll4-KO hEMVs under flow conditions immunostained for <t>VE-cadherin</t> and labeled with phalloidin (actin). c , Quantification of junctional area measured from VE-cadherin immunostained micrographs. d , Immunofluorescent micrographs of recombinant Dll4-HA expressing ECs under static + DMSO, flow + DMSO, and flow + Dynasore conditions stained for HA (Dll4-HA) and DAPI. e , Quantification of internalized Dll4-HA in ECs under static + DMSO, flow + DMSO, and flow + Dynasore conditions. Cells with internalized Dll4-HA counted as those with > 1 AlexaFluor-488 positive puncta. f , Immunofluorescent micrograph of a Dll4-HA expressing EC under flow stained for HA (Dll4-HA), Notch1 ECD, and DAPI. g , Diffusive permeability of 70kDa dextran measured in cells treated with Dynasore hydrate or DMSO load control and exposed to flow overnight. For all plots, mean ± s.e.m., n≥3 hEMVs, **p
    Ve Cadherin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti e cadherin
    Fig. 4. Released E-Cad/CTF2 dissociates from PS1 but remains bound to β-catenin. ( A ) Extracts from STS-treated A431 cells were immunoprecipitated with antibodies against PS1 (I-R222), pre-immune serum (PI-R222), β-catenin or desmoglein, and the immunoprecipitates (IPs) obtained were probed on western blots with <t>anti-E-cadherin</t> antibody C36. For reference, cell lysate was also probed; the asterisk shows IgGs. ( B ) A431 cells treated for 6 h with STS were fractionated into membrane, soluble cytosolic and Triton X-100-insoluble (TX100- insoluble) fractions, and the fractions obtained were then probed on western blots with C36 antibody.
    Anti E Cadherin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 3550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti e cadherin
    Fig. 4. Released E-Cad/CTF2 dissociates from PS1 but remains bound to β-catenin. ( A ) Extracts from STS-treated A431 cells were immunoprecipitated with antibodies against PS1 (I-R222), pre-immune serum (PI-R222), β-catenin or desmoglein, and the immunoprecipitates (IPs) obtained were probed on western blots with <t>anti-E-cadherin</t> antibody C36. For reference, cell lysate was also probed; the asterisk shows IgGs. ( B ) A431 cells treated for 6 h with STS were fractionated into membrane, soluble cytosolic and Triton X-100-insoluble (TX100- insoluble) fractions, and the fractions obtained were then probed on western blots with C36 antibody.
    Anti E Cadherin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti e cadherin
    H. pylori infection of primary-cell preparations and NCI-N87 cells has no effect on IQGAP-1 or <t>E-cadherin</t> protein expression. Ten microliters of H. pylori with an OD 600 of 0.5 was used to inoculate 1 ml of growth medium. The infected epithelial cells were maintained in 10% CO 2 at 37°C for 48 h prior to collection of protein. Whole-cell lysates were prepared from control and infected-cell preparations, and protein was analyzed by SDS-PAGE using 7.5% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were incubated with monoclonal anti-IQGAP-1 and monoclonal <t>anti-E-cadherin</t> antibodies. The density of the bands was related to that of the housekeeping protein histone 2B. Data are representative of three to six independent experiments.
    Mouse Anti E Cadherin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti n cadherin
    H. pylori infection of primary-cell preparations and NCI-N87 cells has no effect on IQGAP-1 or <t>E-cadherin</t> protein expression. Ten microliters of H. pylori with an OD 600 of 0.5 was used to inoculate 1 ml of growth medium. The infected epithelial cells were maintained in 10% CO 2 at 37°C for 48 h prior to collection of protein. Whole-cell lysates were prepared from control and infected-cell preparations, and protein was analyzed by SDS-PAGE using 7.5% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were incubated with monoclonal anti-IQGAP-1 and monoclonal <t>anti-E-cadherin</t> antibodies. The density of the bands was related to that of the housekeeping protein histone 2B. Data are representative of three to six independent experiments.
    Anti N Cadherin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti e cadherin
    Enzymatic activity of cathepsin G is required for cathepsin G-promoted <t>E-cadherin-mediated</t> cell-cell adhesion. MCF-7 cells were incubated in 5% FBS-containing medium on fibronectin for 24 hours. (a) After washing, the adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 24 hours. Cathepsin G-induced cell condensation was observed by phase-contrast microscopy. (b) After washing, adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 3 hours. The immunocomplexes with <t>anti-E-cadherin</t> were then analyzed by immunoblotting with anti-E-cadherin using an anti- β -catenin antibody.
    Anti E Cadherin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2015 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam ve cadherin
    Enzymatic activity of cathepsin G is required for cathepsin G-promoted <t>E-cadherin-mediated</t> cell-cell adhesion. MCF-7 cells were incubated in 5% FBS-containing medium on fibronectin for 24 hours. (a) After washing, the adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 24 hours. Cathepsin G-induced cell condensation was observed by phase-contrast microscopy. (b) After washing, adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 3 hours. The immunocomplexes with <t>anti-E-cadherin</t> were then analyzed by immunoblotting with anti-E-cadherin using an anti- β -catenin antibody.
    Ve Cadherin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti n cadherin
    Expression of Smad3 linker mutant that lacks phosphorylation sites attenuates TGF-β1–induced EMT, migration, and MMP2 activation in A549 lung cancer cells. (A) Schematic representation of Smad3 wild type (S3 WT ) and mutant lacking phosphorylation sites in the linker region (S3 EPSM ). A549 cells infected with lentivirus carrying expression vector for Myc-tagged Smad3-WT (Myc-S3 WT ) or Myc-tagged Smad3-EPSM (Myc-S3 EPSM ), or its corresponding empty vector (pCAG) were stimulated with 5 ng/ml of TGF-β1 for 36 hours and then subjected to Western blot analysis using <t>anti-E-cadherin,</t> <t>anti-N-cadherin,</t> anti-vimentin, and anti-Myc antibodies (B) and immunofluorescence staining for E-cadherin and Myc-S3 EPSM (C). (D) A549 cells (1 × 10 4 ) that were infected as in B were seeded on 8-mm porous Transwell chambers and then stimulated with treatment with 5 ng/ml of TGF-β1 for 36 hours. Transmigrating cells were stained and counted as in Figure 2 B . Quantitative data are shown as the mean ± SD of three independent experiments. ** P
    Anti N Cadherin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ve cadherin
    Stabilization of endothelial <t>VE-cadherin</t> junctions blocks melanoma-induced gap formation and subsequent endothelial barrier breakdown. HPMEC monolayers were treated with FGF1 for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. Results represent the mean +/−SEM, (***p
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    Abcam anti n cadherin antibody
    Stabilization of endothelial <t>VE-cadherin</t> junctions blocks melanoma-induced gap formation and subsequent endothelial barrier breakdown. HPMEC monolayers were treated with FGF1 for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. Results represent the mean +/−SEM, (***p
    Anti N Cadherin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti n cadherin
    Stabilization of endothelial <t>VE-cadherin</t> junctions blocks melanoma-induced gap formation and subsequent endothelial barrier breakdown. HPMEC monolayers were treated with FGF1 for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. Results represent the mean +/−SEM, (***p
    Anti N Cadherin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems n cadherin
    Ngn1 mRNA expression decreases with age; <t>N-cadherin</t> regulates Ngn1 mRNA expression. Panel A shows Ngn1 mRNA expression measured by RT-PCR in young adult and retired breeder neurospheres treated with or without DETA-NONOate for 7 days. Panels B and C show quantitative data of Ngn1 mRNA expression in young neurospheres (B) and retired breeder (C) neurosphere treated with or without DETA-NONOate, N-cadherin, anti-N-cadherin and DETA-NONOate combination with anti-N-cadherin.
    N Cadherin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson ve cadherin
    CD148 strengthens cell-cell adhesion in <t>A431D/E-cadherin</t> WT, but not A431D or A431D/E-cadherin 764AAA, cells. Effects of CD148 in cell-cell adhesion were assessed by a hanging drop assay. Images show representative data of ten independent experiments. CD148 WT, but not CS, remarkably increases the cell-cell adhesion in A431D/E-cadherin WT cells, while it shows no effects in A431D or A431D/E-cadherin 764 AAA cells.
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    Proteintech rabbit n cadherin polyclonal
    CD148 strengthens cell-cell adhesion in <t>A431D/E-cadherin</t> WT, but not A431D or A431D/E-cadherin 764AAA, cells. Effects of CD148 in cell-cell adhesion were assessed by a hanging drop assay. Images show representative data of ten independent experiments. CD148 WT, but not CS, remarkably increases the cell-cell adhesion in A431D/E-cadherin WT cells, while it shows no effects in A431D or A431D/E-cadherin 764 AAA cells.
    Rabbit N Cadherin Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti e cadherin antibody
    CD148 strengthens cell-cell adhesion in <t>A431D/E-cadherin</t> WT, but not A431D or A431D/E-cadherin 764AAA, cells. Effects of CD148 in cell-cell adhesion were assessed by a hanging drop assay. Images show representative data of ten independent experiments. CD148 WT, but not CS, remarkably increases the cell-cell adhesion in A431D/E-cadherin WT cells, while it shows no effects in A431D or A431D/E-cadherin 764 AAA cells.
    Anti E Cadherin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies n cadherin
    Effect of <t>E-cadherin</t> overexpression on cell dimensions. Hearts from adult rabbits were excised and single cells enzymatically isolated using a Langendorff perfusion system. Isolated single adult rabbit cells and H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ Cell dimensions were measured using a graticule in the eye-piece of a microscope. (A) Transduction of H9c2 cells with AD:E-cadherin resulted in significantly decreased cell diameter ( n = 50/group). (B) Infection of rabbit myocytes with Ad:E-cadherin also resulted in cells with a significantly decreased cell length and width resulting in an overall decrease in cell volume ( n = 50/group).
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    Thermo Fisher anti e cadherin
    Effect of <t>E-cadherin</t> overexpression on cell dimensions. Hearts from adult rabbits were excised and single cells enzymatically isolated using a Langendorff perfusion system. Isolated single adult rabbit cells and H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ Cell dimensions were measured using a graticule in the eye-piece of a microscope. (A) Transduction of H9c2 cells with AD:E-cadherin resulted in significantly decreased cell diameter ( n = 50/group). (B) Infection of rabbit myocytes with Ad:E-cadherin also resulted in cells with a significantly decreased cell length and width resulting in an overall decrease in cell volume ( n = 50/group).
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    Image Search Results


    Calcipotriol suppressed the activated HSCs-induced tumor progression of heat-treated residual HCC cells in vivo. a , b The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-exposed residual HCC cells (MHCC97H, Hep3B, HepG2 and Huh7) cultured with conditioned medium (CM) from HSCs or calcipotriol-treated HSCs were compared. HSCs cells (LX2 and pHSC) were stimulated by the pre-treatment with TGF-β1. c The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-treated residual HCC cells were reversed when CM from calcipotriol-treated LX2 cells was supplemented with 100 ng/mL POSTN. d Calcipotriol inhibited tumor growth compared with the control group. The combination therapy of calcipotriol and cisplatin significantly reduced tumor growth. e The expression of POSTN, α-SMA, PCNA, E-Cadherin and cleaved-Caspase-3 was detected by immunohistochemical analysis. Calcipotriol, Cal. ** P

    Journal: Journal of Translational Medicine

    Article Title: Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition

    doi: 10.1186/s12967-018-1676-3

    Figure Lengend Snippet: Calcipotriol suppressed the activated HSCs-induced tumor progression of heat-treated residual HCC cells in vivo. a , b The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-exposed residual HCC cells (MHCC97H, Hep3B, HepG2 and Huh7) cultured with conditioned medium (CM) from HSCs or calcipotriol-treated HSCs were compared. HSCs cells (LX2 and pHSC) were stimulated by the pre-treatment with TGF-β1. c The changes of Snail, Vimentin, E-cadherin and N-cadherin, PCNA expression in heat-treated residual HCC cells were reversed when CM from calcipotriol-treated LX2 cells was supplemented with 100 ng/mL POSTN. d Calcipotriol inhibited tumor growth compared with the control group. The combination therapy of calcipotriol and cisplatin significantly reduced tumor growth. e The expression of POSTN, α-SMA, PCNA, E-Cadherin and cleaved-Caspase-3 was detected by immunohistochemical analysis. Calcipotriol, Cal. ** P

    Article Snippet: Briefly, tissue section was deparaffinised, rehydrated, heated with antigen retrieval, blocked with 3% hydrogen peroxide and then incubated with primary antibody against POSTN (1:200), α-SMA (1:100), PCNA (1:400), cleaved-Caspase-3 (1:100) or E-cadherin (1:100) (Cell Signaling Technology, USA) at 4 °C overnight.

    Techniques: In Vivo, Expressing, Cell Culture, Immunohistochemistry

    Activated HSCs or HSC-CM promoted the proliferation, migration, invasion, colony formation, and decreased the apoptosis of heat-exposed residual HCC cells. a When heat-treated residual HCC cells (Huh7, MHCC97H, Hep3B and HepG2) were co-cultured with activated HSCs LX2, proliferation was significantly higher than the cells cultured alone, as analyzed by WST-1 assay. b , c Proliferation of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells was significantly higher than the cells cultured with control medium, as indicated by the WST-1 assay, BrdU-ELISA and growth curves. d PCNA and cyclinD1 mRNA expression of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells were significantly higher than the cells cultured with control medium, as measured by qRT-PCR. e PCNA protein expression was analyzed by western blot. f Heat-treated residual HCC cells displayed distinct spindle-like appearance when cultured with HSC-CM from LX2 cells. g HSC-CM from LX2 cells promoted the in vitro migration and invasion of heat-treated residual HCC cells. The number of migrated and invaded cells was counted. h Colony formation of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells was increased. i Apoptosis of heat-treated residual HCC cells in the presence of HSC-CM from LX2 cells was reduced than the cell cultured with control medium, as analyzed by Annexin V/PI staining. j – l The levels of Snail mRNA expression, and Snail, Vimentin, E-cadherin, and N-cadherin protein expression in heat-treated residual HCC cells cultured with HSC-CM from LX2 cells or pHSC cells compared with control medium were analyzed by qRT-PCR and western blot. pHSC primary hepatic stellate cells. ** P

    Journal: Journal of Translational Medicine

    Article Title: Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition

    doi: 10.1186/s12967-018-1676-3

    Figure Lengend Snippet: Activated HSCs or HSC-CM promoted the proliferation, migration, invasion, colony formation, and decreased the apoptosis of heat-exposed residual HCC cells. a When heat-treated residual HCC cells (Huh7, MHCC97H, Hep3B and HepG2) were co-cultured with activated HSCs LX2, proliferation was significantly higher than the cells cultured alone, as analyzed by WST-1 assay. b , c Proliferation of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells was significantly higher than the cells cultured with control medium, as indicated by the WST-1 assay, BrdU-ELISA and growth curves. d PCNA and cyclinD1 mRNA expression of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells were significantly higher than the cells cultured with control medium, as measured by qRT-PCR. e PCNA protein expression was analyzed by western blot. f Heat-treated residual HCC cells displayed distinct spindle-like appearance when cultured with HSC-CM from LX2 cells. g HSC-CM from LX2 cells promoted the in vitro migration and invasion of heat-treated residual HCC cells. The number of migrated and invaded cells was counted. h Colony formation of heat-treated residual HCC cells cultured in HSC-CM from LX2 cells was increased. i Apoptosis of heat-treated residual HCC cells in the presence of HSC-CM from LX2 cells was reduced than the cell cultured with control medium, as analyzed by Annexin V/PI staining. j – l The levels of Snail mRNA expression, and Snail, Vimentin, E-cadherin, and N-cadherin protein expression in heat-treated residual HCC cells cultured with HSC-CM from LX2 cells or pHSC cells compared with control medium were analyzed by qRT-PCR and western blot. pHSC primary hepatic stellate cells. ** P

    Article Snippet: Briefly, tissue section was deparaffinised, rehydrated, heated with antigen retrieval, blocked with 3% hydrogen peroxide and then incubated with primary antibody against POSTN (1:200), α-SMA (1:100), PCNA (1:400), cleaved-Caspase-3 (1:100) or E-cadherin (1:100) (Cell Signaling Technology, USA) at 4 °C overnight.

    Techniques: Migration, Cell Culture, WST-1 Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Staining

    POSTN in HSC-CM mediated the increased proliferation, migration, and invasion of heat-exposed residual HCC cells. a , b The alterations of Snail, Vimentin, E-cadherin, N-cadherin, PCNA protein expression in heat-exposed residual HCC cells cultured with POSTN-depleting HSC-CM from LX2 cells or pHSC cells. c Distinct spindle-like appearance of heat-treated residual HCC cells (MHCC97H and HepG2) induced by exogenous POSTN (100 ng/mL). d After treated with exogenous POSTN, heat-exposed residual HepG2 cells showed significantly higher levels of Ki-67, cyclinD1, PCNA, Snail mRNA expression in a dose-dependent manner, as measured by qRT-PCR. e , f After treated with exogenous POSTN, heat-exposed residual HCC cells (Huh7, MHCC97H, HepG2 and Hep3B) showed significantly higher levels of cyclinD1, Snail, PCNA, N-cadherin, Vimentin and markedly lower level of E-cadherin compared to the control, as detected by qRT-PCR and western blot. pHSC primary hepatic stellate cells. ** P

    Journal: Journal of Translational Medicine

    Article Title: Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition

    doi: 10.1186/s12967-018-1676-3

    Figure Lengend Snippet: POSTN in HSC-CM mediated the increased proliferation, migration, and invasion of heat-exposed residual HCC cells. a , b The alterations of Snail, Vimentin, E-cadherin, N-cadherin, PCNA protein expression in heat-exposed residual HCC cells cultured with POSTN-depleting HSC-CM from LX2 cells or pHSC cells. c Distinct spindle-like appearance of heat-treated residual HCC cells (MHCC97H and HepG2) induced by exogenous POSTN (100 ng/mL). d After treated with exogenous POSTN, heat-exposed residual HepG2 cells showed significantly higher levels of Ki-67, cyclinD1, PCNA, Snail mRNA expression in a dose-dependent manner, as measured by qRT-PCR. e , f After treated with exogenous POSTN, heat-exposed residual HCC cells (Huh7, MHCC97H, HepG2 and Hep3B) showed significantly higher levels of cyclinD1, Snail, PCNA, N-cadherin, Vimentin and markedly lower level of E-cadherin compared to the control, as detected by qRT-PCR and western blot. pHSC primary hepatic stellate cells. ** P

    Article Snippet: Briefly, tissue section was deparaffinised, rehydrated, heated with antigen retrieval, blocked with 3% hydrogen peroxide and then incubated with primary antibody against POSTN (1:200), α-SMA (1:100), PCNA (1:400), cleaved-Caspase-3 (1:100) or E-cadherin (1:100) (Cell Signaling Technology, USA) at 4 °C overnight.

    Techniques: Migration, Expressing, Cell Culture, Quantitative RT-PCR, Western Blot

    Activated HSCs promoted the in vivo tumor progression of heat-treated residual HCC cells. a HSCs cells were found in the tumors at 2 weeks after the inoculation of heat-treated residual MHCC97H cells with CFSE-labelled pHSCs. b , c Up-regulation of Ki-67, PCNA, cyclin D1 and Snail mRNA expression was found in the tumors formed from heat-exposed residual MHCC97H and pHSCs compared with the tumors from heat-exposed residual MHCC97H alone, as detected by qRT-PCR. The protein expression of PCNA and E-Cadherin was shown by immunohistochemical analysis. d Xenograft tumorigenicity assay. Heat-exposed residual MHCC97H (2 × 10 4 cells) with 100 ng/mL POSTN subcutaneously inoculated into NOD/SCID mice developed more and larger tumors compared to the cells injected alone. Red arrows on the left flank represent the tumors formed by heat-exposed residual MHCC97H cells with 100 ng/mL POSTN while black arrows on the right flank represent the tumors formed by heat-exposed residual MHCC97H cells alone. The number and images of tumors developed in mice after 8 weeks are shown. The two groups were compared by using Fisher exact test. e The mRNA expression of NANOG, CD133, EpCAM was significantly up-regulated in the tumors generated from heat-exposed residual MHCC97H cells with 100 ng/mL POSTN. ** P

    Journal: Journal of Translational Medicine

    Article Title: Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition

    doi: 10.1186/s12967-018-1676-3

    Figure Lengend Snippet: Activated HSCs promoted the in vivo tumor progression of heat-treated residual HCC cells. a HSCs cells were found in the tumors at 2 weeks after the inoculation of heat-treated residual MHCC97H cells with CFSE-labelled pHSCs. b , c Up-regulation of Ki-67, PCNA, cyclin D1 and Snail mRNA expression was found in the tumors formed from heat-exposed residual MHCC97H and pHSCs compared with the tumors from heat-exposed residual MHCC97H alone, as detected by qRT-PCR. The protein expression of PCNA and E-Cadherin was shown by immunohistochemical analysis. d Xenograft tumorigenicity assay. Heat-exposed residual MHCC97H (2 × 10 4 cells) with 100 ng/mL POSTN subcutaneously inoculated into NOD/SCID mice developed more and larger tumors compared to the cells injected alone. Red arrows on the left flank represent the tumors formed by heat-exposed residual MHCC97H cells with 100 ng/mL POSTN while black arrows on the right flank represent the tumors formed by heat-exposed residual MHCC97H cells alone. The number and images of tumors developed in mice after 8 weeks are shown. The two groups were compared by using Fisher exact test. e The mRNA expression of NANOG, CD133, EpCAM was significantly up-regulated in the tumors generated from heat-exposed residual MHCC97H cells with 100 ng/mL POSTN. ** P

    Article Snippet: Briefly, tissue section was deparaffinised, rehydrated, heated with antigen retrieval, blocked with 3% hydrogen peroxide and then incubated with primary antibody against POSTN (1:200), α-SMA (1:100), PCNA (1:400), cleaved-Caspase-3 (1:100) or E-cadherin (1:100) (Cell Signaling Technology, USA) at 4 °C overnight.

    Techniques: In Vivo, Expressing, Quantitative RT-PCR, Immunohistochemistry, Tumorigenicity Assay, Mouse Assay, Injection, Generated

    POSTN induced the Shc-ERK activation of heat-exposed residual HCC cells through integrin β1. a The mRNA expression profile of heat-treated residual MHCC97H cells in response to POSTN was illustrated as a heatmap. Red, green represent high and low mRNA expression. b With POSTN treatment, the phosphorylated of p52Shc and ERK1/2 in heat-exposed residual HCC cells (MCHCC97H and HepG2) were significantly increased in a time-dependent manner. c PPI network analysis of the differentially expressed genes identified Shc as a gene of biological importance in POSTN-mediated signaling networks and a diagram illustrated the interaction of Shc with the molecules (e.g., ITGB1 and MAPK1). d When heat-exposed residual HCC cells (MCHCC97H and HepG2) were treated with POSTN, the levels of PCNA, N-cadherin and ERK1/2 phosphorylation were significantly increased. ERK1/2 inhibitor (U0126, 25 μM) reversed the above POSTN-induced increase. e With the stimulation of exogenous POSTN, the levels of Ki-67, PCNA and Snail mRNA expression were significantly decreased in heat-exposed residual integrin β1-knockdown MHCC97H cells. f Expression of POSTN in HCC tissues (n = 374) than that of adjacent non-tumor tissues (n = 50) in the HCC data of TCGA cohorts. g A significant positive correlation between the degree of POSTN expression also showed with that of COL1A1 (r = 0.8445, P

    Journal: Journal of Translational Medicine

    Article Title: Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition

    doi: 10.1186/s12967-018-1676-3

    Figure Lengend Snippet: POSTN induced the Shc-ERK activation of heat-exposed residual HCC cells through integrin β1. a The mRNA expression profile of heat-treated residual MHCC97H cells in response to POSTN was illustrated as a heatmap. Red, green represent high and low mRNA expression. b With POSTN treatment, the phosphorylated of p52Shc and ERK1/2 in heat-exposed residual HCC cells (MCHCC97H and HepG2) were significantly increased in a time-dependent manner. c PPI network analysis of the differentially expressed genes identified Shc as a gene of biological importance in POSTN-mediated signaling networks and a diagram illustrated the interaction of Shc with the molecules (e.g., ITGB1 and MAPK1). d When heat-exposed residual HCC cells (MCHCC97H and HepG2) were treated with POSTN, the levels of PCNA, N-cadherin and ERK1/2 phosphorylation were significantly increased. ERK1/2 inhibitor (U0126, 25 μM) reversed the above POSTN-induced increase. e With the stimulation of exogenous POSTN, the levels of Ki-67, PCNA and Snail mRNA expression were significantly decreased in heat-exposed residual integrin β1-knockdown MHCC97H cells. f Expression of POSTN in HCC tissues (n = 374) than that of adjacent non-tumor tissues (n = 50) in the HCC data of TCGA cohorts. g A significant positive correlation between the degree of POSTN expression also showed with that of COL1A1 (r = 0.8445, P

    Article Snippet: Briefly, tissue section was deparaffinised, rehydrated, heated with antigen retrieval, blocked with 3% hydrogen peroxide and then incubated with primary antibody against POSTN (1:200), α-SMA (1:100), PCNA (1:400), cleaved-Caspase-3 (1:100) or E-cadherin (1:100) (Cell Signaling Technology, USA) at 4 °C overnight.

    Techniques: Activation Assay, Expressing

    Effect of Carved on hepatic contents of ( A ) E-Cadherin and ( B ) vimentin. Values are expressed as mean ± S.D (n = 7). Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, P

    Journal: Scientific Reports

    Article Title: Anti-fibrotic impact of Carvedilol in a CCl-4 model of liver fibrosis via serum microRNA-200a/SMAD7 enhancement to bridle TGF-β1/EMT track

    doi: 10.1038/s41598-018-32309-1

    Figure Lengend Snippet: Effect of Carved on hepatic contents of ( A ) E-Cadherin and ( B ) vimentin. Values are expressed as mean ± S.D (n = 7). Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, P

    Article Snippet: Moreover, ELISA kits for E-Cadherin (cat#ab202413), p38MAPK (cat#ER1138), and p -S536-NF-κBp65 (cat#PEL-NFKBP65-S536) were procured from Abcam’s ELISA kit (Cambrige, UK), Fine test (Wuhan, Hubei, China), and Ray Biotech (Georgia, USA), respectively.

    Techniques:

    has/apkc regulates polarised localisation of E-cadherin independent of peridermal cell height. Regression analysis of normalised E-cadherin intensity plotted against normalised cell-height ( A ) performed on data presented in Figure 2A4 . The red line marks the regression line and red shaded area marks the 99% prediction limits as per the sibling data. The points outside the red area are considered to have abnormal distribution. The wild type siblings (WT sib) and has/apkc mutant cells are classified according to the height as tall, medium or short cells ( B ). The apical perimeter was reduced in both tall and medium/short cells ( C1, D1 ) in has/apkc mutant embryos. A graphical representation of E-cadherin localisation along the apicobasal axis ( C2, D2 ) and the noise index ( C3, D3 ) in has/apkc mutants show a decrease in robustness of E-cadherin polarity in both tall ( C2, C3 ) and medium/short cells ( D2, D3 ). Asterisk indicates significant difference (p

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: has/apkc regulates polarised localisation of E-cadherin independent of peridermal cell height. Regression analysis of normalised E-cadherin intensity plotted against normalised cell-height ( A ) performed on data presented in Figure 2A4 . The red line marks the regression line and red shaded area marks the 99% prediction limits as per the sibling data. The points outside the red area are considered to have abnormal distribution. The wild type siblings (WT sib) and has/apkc mutant cells are classified according to the height as tall, medium or short cells ( B ). The apical perimeter was reduced in both tall and medium/short cells ( C1, D1 ) in has/apkc mutant embryos. A graphical representation of E-cadherin localisation along the apicobasal axis ( C2, D2 ) and the noise index ( C3, D3 ) in has/apkc mutants show a decrease in robustness of E-cadherin polarity in both tall ( C2, C3 ) and medium/short cells ( D2, D3 ). Asterisk indicates significant difference (p

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Mutagenesis

    e-cadherin knockdown in the basal epidermis does not have any effect on aPKC localisation in the periderm or basal epidermis. Immunostaining of embryos having Ctrl MO ( A ) and cdh1 MO ( B ) clones marked by GFP ( A1, B1 ) and showing e-cadherin knockdown ( A2, B2 ), aPKC localisation in the basal epidermis ( A3, B3 ) and the apical domain ( A4, B4 ) as well as basal domain of the peridermal cells ( A5, B5 ) lying above the basal epidermal clone. Note that there is no effect on aPKC localisation in the periderm when e-cadherin is knocked down in the basal epidermis. Dotted lines mark the basal epidermal clone and the peridermal region above the clone. Arrowheads (in B2) mark the loss of E-cadherin in the morphant clone. Scale bar equals to 10 µm ( A5, B5 ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: e-cadherin knockdown in the basal epidermis does not have any effect on aPKC localisation in the periderm or basal epidermis. Immunostaining of embryos having Ctrl MO ( A ) and cdh1 MO ( B ) clones marked by GFP ( A1, B1 ) and showing e-cadherin knockdown ( A2, B2 ), aPKC localisation in the basal epidermis ( A3, B3 ) and the apical domain ( A4, B4 ) as well as basal domain of the peridermal cells ( A5, B5 ) lying above the basal epidermal clone. Note that there is no effect on aPKC localisation in the periderm when e-cadherin is knocked down in the basal epidermis. Dotted lines mark the basal epidermal clone and the peridermal region above the clone. Arrowheads (in B2) mark the loss of E-cadherin in the morphant clone. Scale bar equals to 10 µm ( A5, B5 ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Immunostaining, Clone Assay

    Rescue of e-cadherin ( cdh-1 ) morphant phenotype by e-cadherin mRNA. Brightfield image of zebrafish embryos injected with Cdh-1 MO ( A ), Cdh-1 MO with mCherry tagged e-cad mRNA ( B ) and just e-cad-mCherry mRNA ( C ). Insets show a high zoom image of one of the embryos showing its stage; cdh-1 MO embryos are stuck at epiboly ( A’ ), but the cdh-1MO + e-cad mRNA ( B’ ) and e-cad mRNA ( C’ ) embryos cross the epiboly stages. The numbers of embryos from various genotypes at the 70% epiboly stages are quantified in the table ( D ). Arrow shows the initiation of tail bud formation in ( B’, C’ ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Rescue of e-cadherin ( cdh-1 ) morphant phenotype by e-cadherin mRNA. Brightfield image of zebrafish embryos injected with Cdh-1 MO ( A ), Cdh-1 MO with mCherry tagged e-cad mRNA ( B ) and just e-cad-mCherry mRNA ( C ). Insets show a high zoom image of one of the embryos showing its stage; cdh-1 MO embryos are stuck at epiboly ( A’ ), but the cdh-1MO + e-cad mRNA ( B’ ) and e-cad mRNA ( C’ ) embryos cross the epiboly stages. The numbers of embryos from various genotypes at the 70% epiboly stages are quantified in the table ( D ). Arrow shows the initiation of tail bud formation in ( B’, C’ ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Injection

    e-cadherin knockdown results in the loss of β-catenin localisation. Immunostainings of embryos harbouring Ctrl MO ( A ) or cdh-1 MO ( B ) clones marked by GFP ( A1, B1 ) showing E-cadherin ( A2, B2 ) and β-catenin ( A3, B3 ) localisation. Arrow heads ( B2 ) and arrows ( B3 ) mark the loss of E-cadherin and concurrent reduction in β-catenin localisation in morphant cells. Dotted lines demarcate the clones. Scale bar represents 10 µm ( A3, B3 ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: e-cadherin knockdown results in the loss of β-catenin localisation. Immunostainings of embryos harbouring Ctrl MO ( A ) or cdh-1 MO ( B ) clones marked by GFP ( A1, B1 ) showing E-cadherin ( A2, B2 ) and β-catenin ( A3, B3 ) localisation. Arrow heads ( B2 ) and arrows ( B3 ) mark the loss of E-cadherin and concurrent reduction in β-catenin localisation in morphant cells. Dotted lines demarcate the clones. Scale bar represents 10 µm ( A3, B3 ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Clone Assay

    E-cadherin localisation with respect to Lyn-GFP in has/apkc mutants in the periderm. Transverse section through the dorsal head epidermis at 48hpf showing localisation of E-cadherin (magenta) and Lyn-GFP (green) along the apicobasal axis in WT siblings and has/apkc mutants ( A ). Dotted lines in ( A ) mark the base of the epidermis. Confocal micrographs showing localisation of E-cadherin (magenta) and Lyn-GFP (green) along the apicobasal axis (0 µm is apical) in the periderm in WT sibling and has/apkc mutant embryos at 48hpf ( B ). Scale bar represents 10 µm ( A, B ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: E-cadherin localisation with respect to Lyn-GFP in has/apkc mutants in the periderm. Transverse section through the dorsal head epidermis at 48hpf showing localisation of E-cadherin (magenta) and Lyn-GFP (green) along the apicobasal axis in WT siblings and has/apkc mutants ( A ). Dotted lines in ( A ) mark the base of the epidermis. Confocal micrographs showing localisation of E-cadherin (magenta) and Lyn-GFP (green) along the apicobasal axis (0 µm is apical) in the periderm in WT sibling and has/apkc mutant embryos at 48hpf ( B ). Scale bar represents 10 µm ( A, B ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Mutagenesis

    The loss of pen/lgl2 function has no effect on E-cadherin polarity. Immunolocalisation of E-cadherin at various cell heights (0 µm is apical) along the apicobasal axis in wild-type sibling (WT sib) and pen/lgl2 mutant in the periderm ( A1 ) and basal epidermis ( B1 ) at 72hpf. Comparison between WT siblings and pen/lgl2 mutants in the periderm ( A2–A4 ) and the basal epidermis ( B2–B4 ) for height of cells ( A2, B2 ) and apical perimeter ( A3, B3 ). Graphs showing polarised distribution of E-cadherin across normalised cell height in the periderm ( A4 ) and basal epidermis ( B4 ) in WT siblings and pen/lgl2 mutants. Scale bar corresponds to 10 µm in A1 and B1. AU = Arbitrary Units; WT = wild type and sib = sibling/s. Asterisk indicates significant difference (p

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: The loss of pen/lgl2 function has no effect on E-cadherin polarity. Immunolocalisation of E-cadherin at various cell heights (0 µm is apical) along the apicobasal axis in wild-type sibling (WT sib) and pen/lgl2 mutant in the periderm ( A1 ) and basal epidermis ( B1 ) at 72hpf. Comparison between WT siblings and pen/lgl2 mutants in the periderm ( A2–A4 ) and the basal epidermis ( B2–B4 ) for height of cells ( A2, B2 ) and apical perimeter ( A3, B3 ). Graphs showing polarised distribution of E-cadherin across normalised cell height in the periderm ( A4 ) and basal epidermis ( B4 ) in WT siblings and pen/lgl2 mutants. Scale bar corresponds to 10 µm in A1 and B1. AU = Arbitrary Units; WT = wild type and sib = sibling/s. Asterisk indicates significant difference (p

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Mutagenesis

    Model proposing stepwise polarisation of the zebrafish embryonic epidermis. The periderm, polarised at EVL stages, has apically localised aPKC. aPKC controls the polarisation of E-cadherin (E-cad). E-cadherin in one layer controls the localisation of E-cadherin in the other layer and negatively controls the Lgl2 levels at the cortex. Lgl2 further regulates formation of hemidesmosomes- junctions mediating cell-matrix adhesion in the basal epidermis. The polarity is setup in a stepwise manner wherein E-cadherin acts a transducer of polarity between the two layers. The number of cells/boundaries quantified for each figure is compiled as an excel sheet and is available as Figure 6—source data 1 . Details of the numbers of cells, boundaries or clones analysed for each experiment.

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Model proposing stepwise polarisation of the zebrafish embryonic epidermis. The periderm, polarised at EVL stages, has apically localised aPKC. aPKC controls the polarisation of E-cadherin (E-cad). E-cadherin in one layer controls the localisation of E-cadherin in the other layer and negatively controls the Lgl2 levels at the cortex. Lgl2 further regulates formation of hemidesmosomes- junctions mediating cell-matrix adhesion in the basal epidermis. The polarity is setup in a stepwise manner wherein E-cadherin acts a transducer of polarity between the two layers. The number of cells/boundaries quantified for each figure is compiled as an excel sheet and is available as Figure 6—source data 1 . Details of the numbers of cells, boundaries or clones analysed for each experiment.

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Clone Assay

    Localisation of aPKC in the periderm at different developmental stages. Immunolocalisation of aPKC (green) and E-cadherin as well as p63 (basal epidermis marker; both magenta) in the periderm and basal epidermis at 24hpf ( A1, A2 ) and 48hpf ( B1, B2 ) showing absence of aPKC in the basal epidermis. Scale bar in A2 and B2 corresponds to 10 µm.

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Localisation of aPKC in the periderm at different developmental stages. Immunolocalisation of aPKC (green) and E-cadherin as well as p63 (basal epidermis marker; both magenta) in the periderm and basal epidermis at 24hpf ( A1, A2 ) and 48hpf ( B1, B2 ) showing absence of aPKC in the basal epidermis. Scale bar in A2 and B2 corresponds to 10 µm.

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Marker

    Demonstration of polarity defect in has/apkc mutant embryos in live expressing exogenous E-cadherin-mCherry. Localisation of E-cadherin-mCherry (magenta) in the periderm of live wild type (WT) sibling and has/apkc mutant embryos at 48 hpf ( A ). Comparison of heights ( B ) and apical perimeter ( C ) in the periderm of wild-type sibling and has/apkc mutant embryos injected with E-cadherin-mCherry and CAAX-mCherry ( B ) or just E-cadherin-mCherry ( C ). Distribution of normalised E-cadherin intensities plotted against normalised heights in given genotypes ( D ). The stacked bar graph ( E ) shows the percentage of cells showing abnormal polarised distribution in live in wild type (WT) sibling and has/apkc mutant embryos. Scale bar represents 10 µm. WT = wild type; sib = siblings; has/apkc = has/apkc mutants; mut = has/apkc mutants. Source file with fluorescence intensities is available as Figure 2—figure supplement 4—source data 1 . Statistical analysis with p values for all the graphs is available as Figure 2—figure supplement 4—source data 2 . Fluorescence intensities of E-cadherin-mCherry in the periderm of has/apkc mutants and WT siblings imaged live at 48hpf. Statistical summaries of comparisons of cellular attributes and assessment of polarity in has/apkc mutants and WT siblings.

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Demonstration of polarity defect in has/apkc mutant embryos in live expressing exogenous E-cadherin-mCherry. Localisation of E-cadherin-mCherry (magenta) in the periderm of live wild type (WT) sibling and has/apkc mutant embryos at 48 hpf ( A ). Comparison of heights ( B ) and apical perimeter ( C ) in the periderm of wild-type sibling and has/apkc mutant embryos injected with E-cadherin-mCherry and CAAX-mCherry ( B ) or just E-cadherin-mCherry ( C ). Distribution of normalised E-cadherin intensities plotted against normalised heights in given genotypes ( D ). The stacked bar graph ( E ) shows the percentage of cells showing abnormal polarised distribution in live in wild type (WT) sibling and has/apkc mutant embryos. Scale bar represents 10 µm. WT = wild type; sib = siblings; has/apkc = has/apkc mutants; mut = has/apkc mutants. Source file with fluorescence intensities is available as Figure 2—figure supplement 4—source data 1 . Statistical analysis with p values for all the graphs is available as Figure 2—figure supplement 4—source data 2 . Fluorescence intensities of E-cadherin-mCherry in the periderm of has/apkc mutants and WT siblings imaged live at 48hpf. Statistical summaries of comparisons of cellular attributes and assessment of polarity in has/apkc mutants and WT siblings.

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Mutagenesis, Expressing, Injection, Fluorescence

    Quantification of E-cadherin levels in two additional experimental sets to assess the cell autonomous and non-autonomous effect of e -cadherin knockdown. Graphs showing polarised localisation of E-cadherin along normalised cell height in the periderm ( A1, B1, C1, D1 ) and basal epidermis ( A2, B2, C2, D2 ) across various boundaries mentioned upon knockdown of e-cadherin , for two independent experiments ( A1–B2 and C1–D2 ). AU = Arbitrary Units. Source file for fluorescence intensities for the second set of peridermal clones and boundaries analysed in the periderm ( A1 ) and basal epidermis ( A2 ) is available as Figure 4—figure supplement 2—source data 1 and 2 , respectively. Fluorescence intensities for basal epidermal clones in the second set and analysis of boundaries in the periderm ( B1 ) and basal epidermis ( B2 ) are available in Figure 4—figure supplement 2—source data 3 and 4 , respectively. Source file with fluorescence intensities for peridermal clones in the third set and analysis of boundaries in the periderm ( C1 ) and basal epidermis ( C2 ) is available as Figure 4—figure supplement 2—source data 5 and 6 , respectively. Fluorescence intensities for the third set of basal epidermal clones and boundaries analysed in the periderm ( D1 ) and basal epidermis ( D2 ) is available as Figure 4—figure supplement 2—source data 7 and 8 , respectively. Statistical analysis with p values for all the graphs is available as Figure 4—figure supplement 2—source data 9 . Fluorescence intensities of E-cadherin localisation along different boundaries of the peridermal cells when clones are in the periderm for Figure 4—figure supplement 2 A1 (second set). Fluorescence intensities of E-cadherin localisation along different boundaries in the basal epidermis when clones are in the periderm for Figure 4—figure supplement 2 A2 (second set). Fluorescence intensities of E-cadherin localisation along different boundaries in the periderm when clones are in the basal epidermis for Figure 4—figure supplement 2 B1 (second set). Fluorescence intensities of E-cadherin localisation along different boundaries in the basal epidermis when clones are in basal epidermis for Figure 4—figure supplement 2 B2 (second set). Fluorescence intensities of E-cadherin localisation along various boundaries in the periderm when clones are in the periderm for Figure 4—figure supplement 2 C1 (third set). Fluorescence intensities of E-cadherin localisation along different boundaries in the basal epidermis when clones are in the periderm for Figure 4—figure supplement 2 C2 (third set). Fluorescence intensities of E-cadherin across different boundaries in the periderm when clones are in the basal epidermis for Figure 4—figure supplement 2 D1 (third set). Fluorescence intensities of E-cadherin across different boundaries in the basal epidermis when the clones are in the basal epidermis for Figure 4—figure supplement 2 D2 (third set). Statistical summaries of comparisons between E-cadherin levels along cdh1 MO and Ctrl MO boundaries presented in Figure 4—figure supplement 2 A1, A2, B1, B2, C1, C2, D1, D2 .

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Quantification of E-cadherin levels in two additional experimental sets to assess the cell autonomous and non-autonomous effect of e -cadherin knockdown. Graphs showing polarised localisation of E-cadherin along normalised cell height in the periderm ( A1, B1, C1, D1 ) and basal epidermis ( A2, B2, C2, D2 ) across various boundaries mentioned upon knockdown of e-cadherin , for two independent experiments ( A1–B2 and C1–D2 ). AU = Arbitrary Units. Source file for fluorescence intensities for the second set of peridermal clones and boundaries analysed in the periderm ( A1 ) and basal epidermis ( A2 ) is available as Figure 4—figure supplement 2—source data 1 and 2 , respectively. Fluorescence intensities for basal epidermal clones in the second set and analysis of boundaries in the periderm ( B1 ) and basal epidermis ( B2 ) are available in Figure 4—figure supplement 2—source data 3 and 4 , respectively. Source file with fluorescence intensities for peridermal clones in the third set and analysis of boundaries in the periderm ( C1 ) and basal epidermis ( C2 ) is available as Figure 4—figure supplement 2—source data 5 and 6 , respectively. Fluorescence intensities for the third set of basal epidermal clones and boundaries analysed in the periderm ( D1 ) and basal epidermis ( D2 ) is available as Figure 4—figure supplement 2—source data 7 and 8 , respectively. Statistical analysis with p values for all the graphs is available as Figure 4—figure supplement 2—source data 9 . Fluorescence intensities of E-cadherin localisation along different boundaries of the peridermal cells when clones are in the periderm for Figure 4—figure supplement 2 A1 (second set). Fluorescence intensities of E-cadherin localisation along different boundaries in the basal epidermis when clones are in the periderm for Figure 4—figure supplement 2 A2 (second set). Fluorescence intensities of E-cadherin localisation along different boundaries in the periderm when clones are in the basal epidermis for Figure 4—figure supplement 2 B1 (second set). Fluorescence intensities of E-cadherin localisation along different boundaries in the basal epidermis when clones are in basal epidermis for Figure 4—figure supplement 2 B2 (second set). Fluorescence intensities of E-cadherin localisation along various boundaries in the periderm when clones are in the periderm for Figure 4—figure supplement 2 C1 (third set). Fluorescence intensities of E-cadherin localisation along different boundaries in the basal epidermis when clones are in the periderm for Figure 4—figure supplement 2 C2 (third set). Fluorescence intensities of E-cadherin across different boundaries in the periderm when clones are in the basal epidermis for Figure 4—figure supplement 2 D1 (third set). Fluorescence intensities of E-cadherin across different boundaries in the basal epidermis when the clones are in the basal epidermis for Figure 4—figure supplement 2 D2 (third set). Statistical summaries of comparisons between E-cadherin levels along cdh1 MO and Ctrl MO boundaries presented in Figure 4—figure supplement 2 A1, A2, B1, B2, C1, C2, D1, D2 .

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Fluorescence, Clone Assay

    EC1-EC2 domain of E-cadherin is required to maintain Lgl2 levels in the epidermis. Confocal images showing expression and localisation of GFP tagged E-cadherin-FL and ΔEC1-EC2-Ecad (green) ( A1, B1, C1 ) and localisation of E-cadherin (magenta) ( A2, A4, B2, B4, C2 ), Lgl2 ( A3, A5, B3, B5 ) and aPKC ( C3, C4 ). Lgl staining in the periderm ( A3, B5 ) or basal epidermis ( B3, A5 ) is shown as heatmap with calibration bar provided for reference ( A6, B6 ). Arrow shows high levels of Lgl in the ΔEC1-EC2-Ecad expressing cells or juxtaposed cells ( A3, A5, B3, and B5 ). Dotted lines indicate the position of the clone in the periderm ( A1–A3 and C1-C4 ) or basal epidermis ( B1–B3 ) and corresponding region in basal epidermis ( A4–A5 ) or periderm ( B4–B5 ), respectively. Scale bar represents 10 µm in ( A5, B5, C4 ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: EC1-EC2 domain of E-cadherin is required to maintain Lgl2 levels in the epidermis. Confocal images showing expression and localisation of GFP tagged E-cadherin-FL and ΔEC1-EC2-Ecad (green) ( A1, B1, C1 ) and localisation of E-cadherin (magenta) ( A2, A4, B2, B4, C2 ), Lgl2 ( A3, A5, B3, B5 ) and aPKC ( C3, C4 ). Lgl staining in the periderm ( A3, B5 ) or basal epidermis ( B3, A5 ) is shown as heatmap with calibration bar provided for reference ( A6, B6 ). Arrow shows high levels of Lgl in the ΔEC1-EC2-Ecad expressing cells or juxtaposed cells ( A3, A5, B3, and B5 ). Dotted lines indicate the position of the clone in the periderm ( A1–A3 and C1-C4 ) or basal epidermis ( B1–B3 ) and corresponding region in basal epidermis ( A4–A5 ) or periderm ( B4–B5 ), respectively. Scale bar represents 10 µm in ( A5, B5, C4 ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Expressing, Staining

    Localisation of E-cadherin with cell polarity markers in the zebrafish embryonic epidermis. Confocal Z-stack showing localisation of E-cadherin (magenta), Lyn-GFP (green) and aPKC (yellow) in the periderm ( A ). Confocal Z-stacks depicting the apical to basal localisation of Lgl2 (green) and E-cadherin (magenta) in the periderm ( B ) and basal epidermis ( C ) at 48hpf. Transverse section of the epidermis over the eye showing localisation of E-cadherin (magenta) and Lgl2 (green) ( D ). Dotted lines in ( D ) mark the base of the epidermis. 0 µm = apical; Scale bar represents 10 µm ( A, B, C, D ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Localisation of E-cadherin with cell polarity markers in the zebrafish embryonic epidermis. Confocal Z-stack showing localisation of E-cadherin (magenta), Lyn-GFP (green) and aPKC (yellow) in the periderm ( A ). Confocal Z-stacks depicting the apical to basal localisation of Lgl2 (green) and E-cadherin (magenta) in the periderm ( B ) and basal epidermis ( C ) at 48hpf. Transverse section of the epidermis over the eye showing localisation of E-cadherin (magenta) and Lgl2 (green) ( D ). Dotted lines in ( D ) mark the base of the epidermis. 0 µm = apical; Scale bar represents 10 µm ( A, B, C, D ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques:

    e-cadherin knockdown has no effect on localisation of ZO-1. Confocal micrographs of embryos with Ctrl MO ( A ) and cdh1 MO ( B ) clones labelled with GFP ( A1, B1 ) and showing localisation of E-cadherin ( A2, B2 ) and ZO-1 ( A3, B3 ). Note that there is no effect of loss of e-cadherin function on ZO-1 localisation. Dotted lines represent the position of the peridermal clones. Arrowheads mark the reduction in E-cadherin localisation in morphant cells ( B2 ). Scale bar is equivalent to 10 µm ( A3, B3 ).

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: e-cadherin knockdown has no effect on localisation of ZO-1. Confocal micrographs of embryos with Ctrl MO ( A ) and cdh1 MO ( B ) clones labelled with GFP ( A1, B1 ) and showing localisation of E-cadherin ( A2, B2 ) and ZO-1 ( A3, B3 ). Note that there is no effect of loss of e-cadherin function on ZO-1 localisation. Dotted lines represent the position of the peridermal clones. Arrowheads mark the reduction in E-cadherin localisation in morphant cells ( B2 ). Scale bar is equivalent to 10 µm ( A3, B3 ).

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Clone Assay

    Live imaging analysis confirms that E-cadherin localises in a polarised manner in the early developing epidermis. Live confocal analysis ( A ) of E-cadherin-mCherry (magenta) and Lyn-GFP (Green) localisation along the apicobasal axis (0 µm is apical) in the periderm and basal epidermis in wild-type embryos at 48hpf. Quantification of E-cadherin levels along apicobasal axis at the normalised cell heights in the periderm and basal epidermis ( B ). Scale bar equals to 10 µm. Source file with fluorescence intensities for periderm and basal epidermis is available as Figure 1—figure supplement 4—source data 1 and 2 , respectively. Fluorescence intensities of E-cadherin-mCherry in the periderm of live wild type embryos in the background of Lyn-GFP at 48hpf. Fluorescence intensities of E-cadherin-mCherry in the basal epidermis of live wild type embryos in the background of Lyn-GFP at 48hpf.

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Live imaging analysis confirms that E-cadherin localises in a polarised manner in the early developing epidermis. Live confocal analysis ( A ) of E-cadherin-mCherry (magenta) and Lyn-GFP (Green) localisation along the apicobasal axis (0 µm is apical) in the periderm and basal epidermis in wild-type embryos at 48hpf. Quantification of E-cadherin levels along apicobasal axis at the normalised cell heights in the periderm and basal epidermis ( B ). Scale bar equals to 10 µm. Source file with fluorescence intensities for periderm and basal epidermis is available as Figure 1—figure supplement 4—source data 1 and 2 , respectively. Fluorescence intensities of E-cadherin-mCherry in the periderm of live wild type embryos in the background of Lyn-GFP at 48hpf. Fluorescence intensities of E-cadherin-mCherry in the basal epidermis of live wild type embryos in the background of Lyn-GFP at 48hpf.

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Imaging, Fluorescence

    Analysis of co-segregation of 3’- Carboxyfluorescein Standard Morpholino and mCherry; schematic showing classification of boundaries used for E-cadherin quantifications. Morpholino tagged with Carboxyfluorescein ( A2, B2, A5, and B5 ) and mCherry ( A1, B1, A4 and B4 ) showing co-segregation in peridermal clones ( A1–A3 ) but no localisation in basal epidermis ( A4–A6 ). Similarly, clones in basal epidermis showing co-localisation with mCherry ( B4–B6 ) but no localisation in periderm ( B1–B3 ). Arrows point to cells showing co-segregation of labelled morpholino and mCherry. Dotted line in ( A1, A2, A3 ) represents the position of the peridermal clone and the basal epidermal region below the clone ( A4, A5, A6 ). Similarly, dotted lines in ( B4, B5, and B6 ) mark the clones in the basal epidermis and the peridermal region above the clone ( B1, B2, and B3 ). Asterisk marks fluorescence bleed through. Scale bar represents 10 µm ( A6, B6 ). Std MO- Fluor = 3’- Carboxyfluorescein Standard Morpholino. Schematic ( C1, C2 ) showing different types of boundaries defined for quantifications. The two epidermal layers, periderm (blue) and basal epidermis (magenta), overlaid on top of each other having either peridermal clone (green in C1 ) or basal epidermal clone (green in C2 ). Arrowheads demonstrate different types of boundaries namely, clone-clone (CC), Non-clone-Clone (NC), and Non clone-Non clone (NN) for analysing layer autonomous effect and complete overlap (CO), partial overlap (PO), and no overlap (NO) ( C1, C2 ) for analysis of layer non autonomous effect.

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: Analysis of co-segregation of 3’- Carboxyfluorescein Standard Morpholino and mCherry; schematic showing classification of boundaries used for E-cadherin quantifications. Morpholino tagged with Carboxyfluorescein ( A2, B2, A5, and B5 ) and mCherry ( A1, B1, A4 and B4 ) showing co-segregation in peridermal clones ( A1–A3 ) but no localisation in basal epidermis ( A4–A6 ). Similarly, clones in basal epidermis showing co-localisation with mCherry ( B4–B6 ) but no localisation in periderm ( B1–B3 ). Arrows point to cells showing co-segregation of labelled morpholino and mCherry. Dotted line in ( A1, A2, A3 ) represents the position of the peridermal clone and the basal epidermal region below the clone ( A4, A5, A6 ). Similarly, dotted lines in ( B4, B5, and B6 ) mark the clones in the basal epidermis and the peridermal region above the clone ( B1, B2, and B3 ). Asterisk marks fluorescence bleed through. Scale bar represents 10 µm ( A6, B6 ). Std MO- Fluor = 3’- Carboxyfluorescein Standard Morpholino. Schematic ( C1, C2 ) showing different types of boundaries defined for quantifications. The two epidermal layers, periderm (blue) and basal epidermis (magenta), overlaid on top of each other having either peridermal clone (green in C1 ) or basal epidermal clone (green in C2 ). Arrowheads demonstrate different types of boundaries namely, clone-clone (CC), Non-clone-Clone (NC), and Non clone-Non clone (NN) for analysing layer autonomous effect and complete overlap (CO), partial overlap (PO), and no overlap (NO) ( C1, C2 ) for analysis of layer non autonomous effect.

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques: Clone Assay, Fluorescence

    E-cadherin localisation is polarised during early epidermis development. Confocal analysis of E-cadherin (magenta) immunolocalisation along the apicobasal axis (0 µm is apical) in the periderm ( A ) and basal epidermis ( B ) in wild-type embryos from 24hpf to 72hpf. Scale bar equals to 10 µm.

    Journal: eLife

    Article Title: Stepwise polarisation of developing bilayered epidermis is mediated by aPKC and E-cadherin in zebrafish

    doi: 10.7554/eLife.49064

    Figure Lengend Snippet: E-cadherin localisation is polarised during early epidermis development. Confocal analysis of E-cadherin (magenta) immunolocalisation along the apicobasal axis (0 µm is apical) in the periderm ( A ) and basal epidermis ( B ) in wild-type embryos from 24hpf to 72hpf. Scale bar equals to 10 µm.

    Article Snippet: E-cadherin knock down experiments should be better quantified, just like the mutant phenotypes. aPKC, E-cadherin and Lgl2 polarity distributions with reduced levels of E-cadherin should be provided.

    Techniques:

    Different kinetics for disruption of desmosomes and adherens junctions by EGF. (a) SCC 12F cells were treated with EGF for the indicated times, fixed, and then probed with E-cadherin or desmoglein-2 antibodies. Note the relocalization of the desmosomal cadherin desmoglein-2 from the cell borders following treatment with EGF for 4–6 h (white arrows). This pattern differs from that observed for the adherens junctional cadherin, E-cadherin, where strong border staining is evident at 6 hours post EGF treatment (white arrowheads). (b) Cells were treated with EGF, fixed, and then stained with phalloidin to stain the actin cytoskeleton, or with a pan-cytokeratin antibody, to label keratin filaments. Disorganization of the keratin network is seen at 6 hours (white arrow), while the actin cytoskeleton remains intact at 8 hours posttreatment (white arrowheads).

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: Different kinetics for disruption of desmosomes and adherens junctions by EGF. (a) SCC 12F cells were treated with EGF for the indicated times, fixed, and then probed with E-cadherin or desmoglein-2 antibodies. Note the relocalization of the desmosomal cadherin desmoglein-2 from the cell borders following treatment with EGF for 4–6 h (white arrows). This pattern differs from that observed for the adherens junctional cadherin, E-cadherin, where strong border staining is evident at 6 hours post EGF treatment (white arrowheads). (b) Cells were treated with EGF, fixed, and then stained with phalloidin to stain the actin cytoskeleton, or with a pan-cytokeratin antibody, to label keratin filaments. Disorganization of the keratin network is seen at 6 hours (white arrow), while the actin cytoskeleton remains intact at 8 hours posttreatment (white arrowheads).

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques: Staining

    Cadherins do not cointernalize with the EGF receptor. SCC 12F cells were treated with 20 nM EGF for 30 minutes, fixed, and probed for EGF receptor (green) or junctional cadherin (red). White arrows indicate the internalized EGF receptor in the cytoplasm while both junctional cadherins, desmoglein-2 (upper panel), and E-cadherin (lower panel) remain at the cell surface 30 minutes post EGF treatment.

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: Cadherins do not cointernalize with the EGF receptor. SCC 12F cells were treated with 20 nM EGF for 30 minutes, fixed, and probed for EGF receptor (green) or junctional cadherin (red). White arrows indicate the internalized EGF receptor in the cytoplasm while both junctional cadherins, desmoglein-2 (upper panel), and E-cadherin (lower panel) remain at the cell surface 30 minutes post EGF treatment.

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques:

    Effect of MMP inhibition on membrane localization of E-cadherin or desmoglein-2 after EGF treatment. Cells were serum-starved for 24 hours then treated with 50 μ M of the broad spectrum MMP inhibitor GM6001X for 30 minutes before addition of 20 nM EGF for the indicated times. Cells were then fixed and probed with either an E-cadherin antibody that recognizes the intracellular epitope or desmoglein-2 antibody. White arrows indicate the internalized desmoglein-2 even in the presence of inhibitor, while the white arrowheads indicate the retention of E-cadherin at the cell-cell borders.

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: Effect of MMP inhibition on membrane localization of E-cadherin or desmoglein-2 after EGF treatment. Cells were serum-starved for 24 hours then treated with 50 μ M of the broad spectrum MMP inhibitor GM6001X for 30 minutes before addition of 20 nM EGF for the indicated times. Cells were then fixed and probed with either an E-cadherin antibody that recognizes the intracellular epitope or desmoglein-2 antibody. White arrows indicate the internalized desmoglein-2 even in the presence of inhibitor, while the white arrowheads indicate the retention of E-cadherin at the cell-cell borders.

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques: Inhibition

    Desmoglein-2 colocalizes with Rab11, a recycling marker. (a) SCC 12F cells were treated for the indicated times with 20 nM EGF, then fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were stained with antibodies against both desmoglein-2 and rab11. Secondary antibodies tagged with either FITC or Rhodamine were used. Colocalization is detected by the appearance of yellow staining where the red and green overlap, particularly in EGF-treated cells (white arrows). (b) Mandler's overlap coefficient, k1, measures the ratio of cadherin colocalized with Rab11 to total cadherin present.

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: Desmoglein-2 colocalizes with Rab11, a recycling marker. (a) SCC 12F cells were treated for the indicated times with 20 nM EGF, then fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were stained with antibodies against both desmoglein-2 and rab11. Secondary antibodies tagged with either FITC or Rhodamine were used. Colocalization is detected by the appearance of yellow staining where the red and green overlap, particularly in EGF-treated cells (white arrows). (b) Mandler's overlap coefficient, k1, measures the ratio of cadherin colocalized with Rab11 to total cadherin present.

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques: Marker, Staining

    EGF receptor activation leads to accumulation of an E-cadherin extracellular fragment in conditioned medium. Subconfluent cells were serum-starved (without BSA in the medium) for 24 hours. Cells were treated with 20 nM EGF for the indicated times, then the conditioned medium was collected, concentrated, and resolved on an 8% SDS gel. Samples of conditioned media were normalized to the corresponding protein lysate, transferred onto PVDF, and probed with an antibody against the extracellular epitope of E-cadherin, which recognizes both full length (120 kD) and cleaved E-cadherin (80 kD).

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: EGF receptor activation leads to accumulation of an E-cadherin extracellular fragment in conditioned medium. Subconfluent cells were serum-starved (without BSA in the medium) for 24 hours. Cells were treated with 20 nM EGF for the indicated times, then the conditioned medium was collected, concentrated, and resolved on an 8% SDS gel. Samples of conditioned media were normalized to the corresponding protein lysate, transferred onto PVDF, and probed with an antibody against the extracellular epitope of E-cadherin, which recognizes both full length (120 kD) and cleaved E-cadherin (80 kD).

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques: Activation Assay, SDS-Gel

    EGF regulation of cadherin proteins in membrane and cytoskeleton-associated pools. Cells were grown to subconfluence, placed in serum-free medium overnight, and then treated with EGF for the indicated times. DG2: desmoglein-2, EC: E-cadherin, AC: alpha-catenin. (a) Sequential detergent extraction separates the triton soluble (membrane-associated) fraction, from the triton insoluble, (cytoskeletal, or intact junctional) fraction. Blots are representative of a minimum of three separate experiments. Bar graphs represent the densitometric quantification of each lane normalized to no treatment control, with asterisks indicating statistical significance. ( P

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: EGF regulation of cadherin proteins in membrane and cytoskeleton-associated pools. Cells were grown to subconfluence, placed in serum-free medium overnight, and then treated with EGF for the indicated times. DG2: desmoglein-2, EC: E-cadherin, AC: alpha-catenin. (a) Sequential detergent extraction separates the triton soluble (membrane-associated) fraction, from the triton insoluble, (cytoskeletal, or intact junctional) fraction. Blots are representative of a minimum of three separate experiments. Bar graphs represent the densitometric quantification of each lane normalized to no treatment control, with asterisks indicating statistical significance. ( P

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques:

    Cadherin staining 48 h postincisional wounding in vivo . Left Panel: E-cadherin immunoreactivity at wound margin 48 h after introduction of an incisional wound. Bar = 150 μ m. Inset: Higher magnification of the advancing edge of epithelium. Note that E-cadherin immunoreactivity is present in all but a few cells at the very tip of the migrating epithelium. Bar = 50 μ m. Right Panel: Desmoglein immunoreactivity at wound margin 48 h after introduction of an incisional wound. Bar = 150 μ m. Inset: Higher magnification of the advancing edge of epithelium. Note that desmoglein immunoreactivity is largely absent from the tip of the migrating epithelium. Bar = 50 μ m.

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: Cadherin staining 48 h postincisional wounding in vivo . Left Panel: E-cadherin immunoreactivity at wound margin 48 h after introduction of an incisional wound. Bar = 150 μ m. Inset: Higher magnification of the advancing edge of epithelium. Note that E-cadherin immunoreactivity is present in all but a few cells at the very tip of the migrating epithelium. Bar = 50 μ m. Right Panel: Desmoglein immunoreactivity at wound margin 48 h after introduction of an incisional wound. Bar = 150 μ m. Inset: Higher magnification of the advancing edge of epithelium. Note that desmoglein immunoreactivity is largely absent from the tip of the migrating epithelium. Bar = 50 μ m.

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques: Staining, In Vivo

    Desmoglein-2 does not internalize with E-cadherin. SCC 12F cells were treated with EGF for the indicated times, fixed, then probed with E-cadherin or desmoglein-2 antibodies. The desmosomal cadherin desmoglein-2 is relocalized from the cell borders at 6–8 hrs after EGF treatment. Note punctate cytoplasmic staining (white arrows). This pattern differs from that observed for the adherens junctional cadherin, E-cadherin, where strong border staining is evident at 6 hours post EGF treatment.

    Journal: Dermatology Research and Practice

    Article Title: Differential Downregulation of E-Cadherin and Desmoglein by Epidermal Growth Factor

    doi: 10.1155/2012/309587

    Figure Lengend Snippet: Desmoglein-2 does not internalize with E-cadherin. SCC 12F cells were treated with EGF for the indicated times, fixed, then probed with E-cadherin or desmoglein-2 antibodies. The desmosomal cadherin desmoglein-2 is relocalized from the cell borders at 6–8 hrs after EGF treatment. Note punctate cytoplasmic staining (white arrows). This pattern differs from that observed for the adherens junctional cadherin, E-cadherin, where strong border staining is evident at 6 hours post EGF treatment.

    Article Snippet: Wound Margins In Vivo Show a Decrease in E-Cadherin and Desmoglein at the Migrating Epithelial Tip Both E-cadherin and desmoglein were studied in an in vivo incisional wound model. After 48 hours after incision, the epithelium stained for both E-cadherin ( , left panel) and desmoglein ( , right panel), throughout a majority of the intact epithelium.

    Techniques: Staining

    Notch1 is activated in response to shear stress by endocytosis of Dll4 a. ICD cleavage as measured by western blot with an antibody specific to cleaved ICD (N1 V1754) in Scramble and Dll4-KO ECs under flow. b , Fluorescent micrographs of Scramble and Dll4-KO hEMVs under flow conditions immunostained for VE-cadherin and labeled with phalloidin (actin). c , Quantification of junctional area measured from VE-cadherin immunostained micrographs. d , Immunofluorescent micrographs of recombinant Dll4-HA expressing ECs under static + DMSO, flow + DMSO, and flow + Dynasore conditions stained for HA (Dll4-HA) and DAPI. e , Quantification of internalized Dll4-HA in ECs under static + DMSO, flow + DMSO, and flow + Dynasore conditions. Cells with internalized Dll4-HA counted as those with > 1 AlexaFluor-488 positive puncta. f , Immunofluorescent micrograph of a Dll4-HA expressing EC under flow stained for HA (Dll4-HA), Notch1 ECD, and DAPI. g , Diffusive permeability of 70kDa dextran measured in cells treated with Dynasore hydrate or DMSO load control and exposed to flow overnight. For all plots, mean ± s.e.m., n≥3 hEMVs, **p

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: Notch1 is activated in response to shear stress by endocytosis of Dll4 a. ICD cleavage as measured by western blot with an antibody specific to cleaved ICD (N1 V1754) in Scramble and Dll4-KO ECs under flow. b , Fluorescent micrographs of Scramble and Dll4-KO hEMVs under flow conditions immunostained for VE-cadherin and labeled with phalloidin (actin). c , Quantification of junctional area measured from VE-cadherin immunostained micrographs. d , Immunofluorescent micrographs of recombinant Dll4-HA expressing ECs under static + DMSO, flow + DMSO, and flow + Dynasore conditions stained for HA (Dll4-HA) and DAPI. e , Quantification of internalized Dll4-HA in ECs under static + DMSO, flow + DMSO, and flow + Dynasore conditions. Cells with internalized Dll4-HA counted as those with > 1 AlexaFluor-488 positive puncta. f , Immunofluorescent micrograph of a Dll4-HA expressing EC under flow stained for HA (Dll4-HA), Notch1 ECD, and DAPI. g , Diffusive permeability of 70kDa dextran measured in cells treated with Dynasore hydrate or DMSO load control and exposed to flow overnight. For all plots, mean ± s.e.m., n≥3 hEMVs, **p

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Western Blot, Flow Cytometry, Labeling, Recombinant, Expressing, Staining, Permeability

    The Notch1 mechanosensory complex stabilizes cell-cell junctions through activation of Rac1 Flow induces endocytosis of Dll4, triggering the activation and cleavage of Notch1 ICD and ECD, which allows the N1-TMD domain to scaffold the adaptor protein LAR with VE-cadherin and recruit the Rac1 GEF Trio to AJs. The resulting complex activates Rac1, elaborates cortical actin, and stabilizes cell-cell junctions to establish barrier function.

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: The Notch1 mechanosensory complex stabilizes cell-cell junctions through activation of Rac1 Flow induces endocytosis of Dll4, triggering the activation and cleavage of Notch1 ICD and ECD, which allows the N1-TMD domain to scaffold the adaptor protein LAR with VE-cadherin and recruit the Rac1 GEF Trio to AJs. The resulting complex activates Rac1, elaborates cortical actin, and stabilizes cell-cell junctions to establish barrier function.

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Activation Assay, Flow Cytometry

    The Notch1 transmembrane domain mediates barrier function through interaction with VE-cadherin a , A library of endogenous Notch1 truncation mutants and over-expression rescue constructs were generated to examine the key functional domains of Notch1 that regulate barrier function. b , P D for ECs with CRISPR/Cas9-mediated endogenous truncation of Notch1 ICD (ICD-KO) or truncation of the TMD and ICD (TMD-ICD-KO) cultured statically, under flow, or in the presence of rDll4-coated collagen. c , Fluorescent micrographs of VE-cadherin and actin for ICD-KO and TMD-ICD-KO ECs under flow conditions. d , Quantification of junctional area measured from VE-cadherin immunostained micrographs. e , P D for N1-KO ECs expressing TMD-ICD-mApple, TMD-ICD V1754G-mApple, TMD-mApple, or mApple infection control cultured statically, under flow, or in the presence of rDll4-coated collagen. f , Fluorescent micrographs of VE-cadherin (magenta), actin (green), and DAPI (blue) in static N1-KO cells expressing TMD-mApple or mApple infection control. g , Quantification of junctional area measured from VE-cadherin immunostained micrographs. h , P D for static N1-KO cells expressing TMD-ICD-mApple or ICD-mApple exposed to DAPT or DMSO load control. i , Immunofluorescent images of Notch1-KO cells expressing either mApple or TMD-mApple, co-stained for VE-cadherin. Co-localization of Notch1 TMD and VE-cadherin (red arrow) is lost at free edges (blue arrow). j , Immunoprecipitation of VE-cadherin and N-Cadherin from Notch1-KO cells expressing either mApple or TMD-mApple. Immunoblotting with a RFP antibody identified co-immunoprecipiting TMD-mApple. For all plots, mean ± s.e.m., n≥3 independent hEMVs, *p

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: The Notch1 transmembrane domain mediates barrier function through interaction with VE-cadherin a , A library of endogenous Notch1 truncation mutants and over-expression rescue constructs were generated to examine the key functional domains of Notch1 that regulate barrier function. b , P D for ECs with CRISPR/Cas9-mediated endogenous truncation of Notch1 ICD (ICD-KO) or truncation of the TMD and ICD (TMD-ICD-KO) cultured statically, under flow, or in the presence of rDll4-coated collagen. c , Fluorescent micrographs of VE-cadherin and actin for ICD-KO and TMD-ICD-KO ECs under flow conditions. d , Quantification of junctional area measured from VE-cadherin immunostained micrographs. e , P D for N1-KO ECs expressing TMD-ICD-mApple, TMD-ICD V1754G-mApple, TMD-mApple, or mApple infection control cultured statically, under flow, or in the presence of rDll4-coated collagen. f , Fluorescent micrographs of VE-cadherin (magenta), actin (green), and DAPI (blue) in static N1-KO cells expressing TMD-mApple or mApple infection control. g , Quantification of junctional area measured from VE-cadherin immunostained micrographs. h , P D for static N1-KO cells expressing TMD-ICD-mApple or ICD-mApple exposed to DAPT or DMSO load control. i , Immunofluorescent images of Notch1-KO cells expressing either mApple or TMD-mApple, co-stained for VE-cadherin. Co-localization of Notch1 TMD and VE-cadherin (red arrow) is lost at free edges (blue arrow). j , Immunoprecipitation of VE-cadherin and N-Cadherin from Notch1-KO cells expressing either mApple or TMD-mApple. Immunoblotting with a RFP antibody identified co-immunoprecipiting TMD-mApple. For all plots, mean ± s.e.m., n≥3 independent hEMVs, *p

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Over Expression, Construct, Generated, Functional Assay, CRISPR, Cell Culture, Flow Cytometry, Expressing, Infection, Staining, Immunoprecipitation

    Non-transcriptional Notch1 signaling regulates vascular barrier function a , Gene expression of Notch1 target genes HES1 and HEY1, the Notch1 ligand DLL4, and VE-cadherin (CDH5) measured via qPCR in ECs treated with DAPT or DMSO load control on Dll4-coated and control tissue culture plastic substrates. b , Fluorescent micrograph of rDll4-coated device prior to cell seeding (green – Alexa Fluor 488 Collagen I, red – immunostain of Dll4). c , Fluorescent micrograph of ECs in hEMVs coated with rDll4 prior to cell seeding. d , Micrographs of GFP-infection control cells under flow. e , Gene expression of HES1, HEY1, DLL4, NOTCH1, and VE-cadherin (CDH5) measured via qPCR in ECs expressing dnMAML or infection control (GFP). f , Western blot validation of Notch1 CRISPR lines: Scramble, Notch1-KO, TMD+ICD-KO, and ICD-KO. g , Fluorescent micrographs of CRISPR/Cas9 scramble control cells under flow. h , Fluorescent micrographs of Scramble and Notch1-KO hEMVs under static conditions immunostained for VE-cadherin and labeled with phalloidin (actin). i , Quantification of junctional area measured from VE-cadherin immunostained micrographs. j , Gene expression measured via qPCR in Notch1-KO cells and scramble control cells. k , Quantification of cell number in f.o.v at 10x magnification of Scramble or Notch1-KO hEMV under static and flow conditions. l , Micrographs of nuclei as visualized by DAPI in Scramble or Notch1-KO hEMVs. For all plots, mean ± s.e.m., n≥3 hEMVs, **p

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: Non-transcriptional Notch1 signaling regulates vascular barrier function a , Gene expression of Notch1 target genes HES1 and HEY1, the Notch1 ligand DLL4, and VE-cadherin (CDH5) measured via qPCR in ECs treated with DAPT or DMSO load control on Dll4-coated and control tissue culture plastic substrates. b , Fluorescent micrograph of rDll4-coated device prior to cell seeding (green – Alexa Fluor 488 Collagen I, red – immunostain of Dll4). c , Fluorescent micrograph of ECs in hEMVs coated with rDll4 prior to cell seeding. d , Micrographs of GFP-infection control cells under flow. e , Gene expression of HES1, HEY1, DLL4, NOTCH1, and VE-cadherin (CDH5) measured via qPCR in ECs expressing dnMAML or infection control (GFP). f , Western blot validation of Notch1 CRISPR lines: Scramble, Notch1-KO, TMD+ICD-KO, and ICD-KO. g , Fluorescent micrographs of CRISPR/Cas9 scramble control cells under flow. h , Fluorescent micrographs of Scramble and Notch1-KO hEMVs under static conditions immunostained for VE-cadherin and labeled with phalloidin (actin). i , Quantification of junctional area measured from VE-cadherin immunostained micrographs. j , Gene expression measured via qPCR in Notch1-KO cells and scramble control cells. k , Quantification of cell number in f.o.v at 10x magnification of Scramble or Notch1-KO hEMV under static and flow conditions. l , Micrographs of nuclei as visualized by DAPI in Scramble or Notch1-KO hEMVs. For all plots, mean ± s.e.m., n≥3 hEMVs, **p

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, Flow Cytometry, Western Blot, CRISPR, Labeling

    Notch1 regulates junctional stability through association with VE-cadherin a , Timelapse images of cells expressing VE-cadherin-mApple in the presence of DAPT or DMSO load control demonstrate that AJs disassemble after 30min of exposure to DAPT, leading to macroscopic intercellular gaps (red arrows). b , Fluorescent micrographs of Notch1 KO cells expressing TMD+ICD-mApple or TMD+ICD V1754G-mApple immunostained for cleaved Notch1 ICD V1754 and DAPI. c , Fluorescent micrographs of TMD-mApple expressed in VE-cadherin knockout or scramble control ECs and immunostained for VE-cadherin. d , Western blot for Notch1 ICD and VE-cadherin in Notch1-KO and VE-cadherin KO ECs. e , Western blot validation of Notch1 rescue constructs: mApple, TMD-mApple, ICD+TMD-mApple, and ICD+TMD V1754G-mApple. f , Immunoprecipitation of VE-cadherin from hMVEC-D cells treated with DMSO or DAPT. Co-immunoprecipitation of mechanosensory complex components was assessed by immunoblotting for Notch1 ICD, Trio, and LAR. g. Western blot of VE-cadherin immunoprecipitations from Notch1 KO cells expressing Notch1-TMD truncation constructs (6, 8, 12 amino acids from the N-terminus) fused to mApple. h , Western blot of VE-cadherin immunoprecipitations from Notch1 KO cells expressing single and dual point mutation Notch1-TMD constructs (within the transmembrane segment of Notch1 TMD) fused to mApple. All images representative of at least three independent experiments.

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: Notch1 regulates junctional stability through association with VE-cadherin a , Timelapse images of cells expressing VE-cadherin-mApple in the presence of DAPT or DMSO load control demonstrate that AJs disassemble after 30min of exposure to DAPT, leading to macroscopic intercellular gaps (red arrows). b , Fluorescent micrographs of Notch1 KO cells expressing TMD+ICD-mApple or TMD+ICD V1754G-mApple immunostained for cleaved Notch1 ICD V1754 and DAPI. c , Fluorescent micrographs of TMD-mApple expressed in VE-cadherin knockout or scramble control ECs and immunostained for VE-cadherin. d , Western blot for Notch1 ICD and VE-cadherin in Notch1-KO and VE-cadherin KO ECs. e , Western blot validation of Notch1 rescue constructs: mApple, TMD-mApple, ICD+TMD-mApple, and ICD+TMD V1754G-mApple. f , Immunoprecipitation of VE-cadherin from hMVEC-D cells treated with DMSO or DAPT. Co-immunoprecipitation of mechanosensory complex components was assessed by immunoblotting for Notch1 ICD, Trio, and LAR. g. Western blot of VE-cadherin immunoprecipitations from Notch1 KO cells expressing Notch1-TMD truncation constructs (6, 8, 12 amino acids from the N-terminus) fused to mApple. h , Western blot of VE-cadherin immunoprecipitations from Notch1 KO cells expressing single and dual point mutation Notch1-TMD constructs (within the transmembrane segment of Notch1 TMD) fused to mApple. All images representative of at least three independent experiments.

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Expressing, Knock-Out, Western Blot, Construct, Immunoprecipitation, Mutagenesis

    Notch1 regulates shear stress-induced vascular barrier function a , Organotypic microfluidic devices of human engineered microvessels (hEMVs) consisting of human ECs (red) in physiologic ECM (green), enabling vessel perfusion at a defined luminal shear stress (inlet and outlet in blue). Inset: 3D reconstruction of hEMVs (red - 70kDa dextran, white – VE-cadherin, green – collagen I). b , Real-time assessment of vascular barrier function in hEMVs cultured statically or under flow (heatmap of fluorescent intensity of 70 kDa dextran, imaging plane indicated by dashed line in a ). c , Quantification of the diffusive permeability (P D ) of 70 kDa dextran across EC barrier as a function of endothelial wall shear stress. d , Relative gene expression for ECs cultured statically or under flow was quantified with qPCR, and Notch target genes regulated by flow are indicated (each column representative of an independent experiment, full gene panel in Extended Data Fig. 2 ). e , ICD cleavage in static and flow EC lysates as measured by western blot with an antibody specific to cleaved ICD (N1 V1754). f , P D measured in hEMVs under static or flow conditions in the presence of Notch inhibitor (DAPT) or rDll4-coated collagen (rDll4). g , Fluorescent micrographs of hEMVs immunostained for VE-cadherin (magenta) and labeled with phalloidin (actin - green) and DAPI (nucleus – blue). h , Quantification of junctional area measured from VE-cadherin immunostained micrographs. i , P D for hEMVs cultured statically, under flow, or in the presence of rDll4-coated collagen with ECs expressing dnMAML, GFP infection control, or with N1-KO, Dll4-KO, or scramble control ECs. j , Fluorescent micrographs of actin and VE-cadherin for hEMVs under flow with ECs expressing dnMAML or with N1-KO ECs. k , Quantification of junctional area measured from VE-cadherin immunostained micrographs (micrographs of GFP and scramble controls in Extended Data Fig. 3 ). For ( a - k ), n≥3 independent hEMVs, mean ± s.e.m. l , Fluorescence intensity heatmaps of Evan’s blue (EB) dye in the mouse dermal vasculature 30min and 60min after IV co-injection of EB and DAPT or DMSO vehicle control. m , Quantification of P D for EB diffusion into the dermal interstitial space (n = 15 vessels across 3 mice/condition). n , Color image of lungs harvested from mice sacrificed 30 min after IV injection of EB. o , Vascular permeability was quantified by eluting and measuring the concentration of EB in lungs relative to the blood EB concentration, (n=4 age, sex-matched littermates, color coded, two-way ANOVA). p , Whole-mount lung vasculature immunostains showing leaked EB and DAPI. *p

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: Notch1 regulates shear stress-induced vascular barrier function a , Organotypic microfluidic devices of human engineered microvessels (hEMVs) consisting of human ECs (red) in physiologic ECM (green), enabling vessel perfusion at a defined luminal shear stress (inlet and outlet in blue). Inset: 3D reconstruction of hEMVs (red - 70kDa dextran, white – VE-cadherin, green – collagen I). b , Real-time assessment of vascular barrier function in hEMVs cultured statically or under flow (heatmap of fluorescent intensity of 70 kDa dextran, imaging plane indicated by dashed line in a ). c , Quantification of the diffusive permeability (P D ) of 70 kDa dextran across EC barrier as a function of endothelial wall shear stress. d , Relative gene expression for ECs cultured statically or under flow was quantified with qPCR, and Notch target genes regulated by flow are indicated (each column representative of an independent experiment, full gene panel in Extended Data Fig. 2 ). e , ICD cleavage in static and flow EC lysates as measured by western blot with an antibody specific to cleaved ICD (N1 V1754). f , P D measured in hEMVs under static or flow conditions in the presence of Notch inhibitor (DAPT) or rDll4-coated collagen (rDll4). g , Fluorescent micrographs of hEMVs immunostained for VE-cadherin (magenta) and labeled with phalloidin (actin - green) and DAPI (nucleus – blue). h , Quantification of junctional area measured from VE-cadherin immunostained micrographs. i , P D for hEMVs cultured statically, under flow, or in the presence of rDll4-coated collagen with ECs expressing dnMAML, GFP infection control, or with N1-KO, Dll4-KO, or scramble control ECs. j , Fluorescent micrographs of actin and VE-cadherin for hEMVs under flow with ECs expressing dnMAML or with N1-KO ECs. k , Quantification of junctional area measured from VE-cadherin immunostained micrographs (micrographs of GFP and scramble controls in Extended Data Fig. 3 ). For ( a - k ), n≥3 independent hEMVs, mean ± s.e.m. l , Fluorescence intensity heatmaps of Evan’s blue (EB) dye in the mouse dermal vasculature 30min and 60min after IV co-injection of EB and DAPT or DMSO vehicle control. m , Quantification of P D for EB diffusion into the dermal interstitial space (n = 15 vessels across 3 mice/condition). n , Color image of lungs harvested from mice sacrificed 30 min after IV injection of EB. o , Vascular permeability was quantified by eluting and measuring the concentration of EB in lungs relative to the blood EB concentration, (n=4 age, sex-matched littermates, color coded, two-way ANOVA). p , Whole-mount lung vasculature immunostains showing leaked EB and DAPI. *p

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Cell Culture, Flow Cytometry, Imaging, Permeability, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Labeling, Infection, Fluorescence, Injection, Diffusion-based Assay, Mouse Assay, IV Injection, Concentration Assay

    Notch1 assembles a mechanosensory junctional complex involving LAR, Trio, and Rac a , Fluorescent micrographs of phalloidin stained ECs. b , Intensity of cortical actin at cell-cell junctions and the number of stress fibers per micron quantified from phalloidin stained ECs (n=3 hEMVs). c , Active Rac1 was isolated from scramble and Notch1-KO cell lysates using a recombinant p21-binding domain of Pak1 (GST-PBD). d , Quantification of western blot band intensity revealed a decrease in Rac1 (~50%) after knockout of Notch1 (n=3 independent lysates). e , Active Rac1 was isolated using GST-PBD from Notch1-KO cell lysates expressing mApple or TMD-mApple. f , Quantification of western blot band intensity revealed an increase in Rac1 (~45%) with expression of TMD-mApple (n=3 independent lysates). g , P D of Notch1-KO ECs expressing TMD-mApple or mApple control in the presence of 50 µM NSC 23766, a Rac1 inhibitor, or vehicle control. h , Immunoprecipitation of VE-cadherin and the Rac1 GEF Trio from ECs cultured statically or under flow. Co-immunoprecipitation of mechanosensory complex proteins was assessed by immunoblotting for Notch1-ECD, Notch1-ICD, LAR (85 kDa P-subunit), and VE-cadherin. i , Immunoprecipitation of VE-cadherin or Trio from scramble and Notch1-KO ECS cultured under flow. Co-immunoprecipitation of mechanosensory complex constituents was assessed by immunoblotting for VE-cadherin, LAR, and Trio. j , P D of LAR-KO and Trio-KO ECs cultured under static or flow conditions or in the presence of rDll4-coated collagen. k , Micrographs of Trio-KO and LAR-KO cells under flow conditions (VE-cadherin – magenta, actin – green, DAPI – blue). l , Quantification of junctional area measured from VE-cadherin immunostained micrographs. m , Immunoprecipitation of VE-cadherin and Trio from Notch1-KO cells expressing TMD-mApple or mApple. Immunoblotting with LAR, VE-cadherin, and RFP antibodies was used to assess co-immunoprecipitation of mechanosensory complex upon expression of TMD. n. P D for scramble, N1KO, LAR-KO, and Trio-KO cells cultured under static conditions expressing TMD-mApple or mApple infection control. For ( g,j,n) , n≥3 hEMVs. All plots mean ± s.e.m., *p

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: Notch1 assembles a mechanosensory junctional complex involving LAR, Trio, and Rac a , Fluorescent micrographs of phalloidin stained ECs. b , Intensity of cortical actin at cell-cell junctions and the number of stress fibers per micron quantified from phalloidin stained ECs (n=3 hEMVs). c , Active Rac1 was isolated from scramble and Notch1-KO cell lysates using a recombinant p21-binding domain of Pak1 (GST-PBD). d , Quantification of western blot band intensity revealed a decrease in Rac1 (~50%) after knockout of Notch1 (n=3 independent lysates). e , Active Rac1 was isolated using GST-PBD from Notch1-KO cell lysates expressing mApple or TMD-mApple. f , Quantification of western blot band intensity revealed an increase in Rac1 (~45%) with expression of TMD-mApple (n=3 independent lysates). g , P D of Notch1-KO ECs expressing TMD-mApple or mApple control in the presence of 50 µM NSC 23766, a Rac1 inhibitor, or vehicle control. h , Immunoprecipitation of VE-cadherin and the Rac1 GEF Trio from ECs cultured statically or under flow. Co-immunoprecipitation of mechanosensory complex proteins was assessed by immunoblotting for Notch1-ECD, Notch1-ICD, LAR (85 kDa P-subunit), and VE-cadherin. i , Immunoprecipitation of VE-cadherin or Trio from scramble and Notch1-KO ECS cultured under flow. Co-immunoprecipitation of mechanosensory complex constituents was assessed by immunoblotting for VE-cadherin, LAR, and Trio. j , P D of LAR-KO and Trio-KO ECs cultured under static or flow conditions or in the presence of rDll4-coated collagen. k , Micrographs of Trio-KO and LAR-KO cells under flow conditions (VE-cadherin – magenta, actin – green, DAPI – blue). l , Quantification of junctional area measured from VE-cadherin immunostained micrographs. m , Immunoprecipitation of VE-cadherin and Trio from Notch1-KO cells expressing TMD-mApple or mApple. Immunoblotting with LAR, VE-cadherin, and RFP antibodies was used to assess co-immunoprecipitation of mechanosensory complex upon expression of TMD. n. P D for scramble, N1KO, LAR-KO, and Trio-KO cells cultured under static conditions expressing TMD-mApple or mApple infection control. For ( g,j,n) , n≥3 hEMVs. All plots mean ± s.e.m., *p

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Staining, Isolation, Recombinant, Binding Assay, Western Blot, Knock-Out, Expressing, Immunoprecipitation, Cell Culture, Flow Cytometry, Infection

    Notch1 regulates VE-cadherin interacting proteins to form the Notch1 mechanosensory complex a , Immunoprecipitation of VE-cadherin from scramble and Notch1-KO cells. Co-immunoprecipitation of candidate Notch1-dependent, VE-cadherin effectors was assessed by immunoblotting for VE-PTP, VEGFR2, and LAR (85 kDa P-subunit). b , Immunoprecipitation of the Rac1 GEF Trio from scramble, Notch1-KO, and LAR-KO cells. Immunoblotting for VE-cadherin was used to assess impaired Trio-VE-cadherin co-immunoprecipitation upon depletion of Notch1 or LAR. c , Western blots of VE-cadherin immunoprecipitated from the lysates of lungs from CDH5-Cre(+); NOTCH1 fl/fl and control CDH5-Cre(+); NOTCH1 fl/fl mice and immunoblotted for LAR. d , Western blots of Trio immunoprecipitated from the lysates of lungs from CDH5-Cre(+); NOTCH1 fl/fl and control CDH5-Cre(+); NOTCH1 fl/fl mice and immunoblotted for VE-cadherin. e , Western blot of proximal interacting proteins extracted with streptavidin from hMVEC-D cells expressing BirA-HA (BioID) or VE-cadherin-BirA-HA (VE-BioID) that were treated with DMSO, Dll4, or DAPT, immunoblotted for HA and Notch1 ICD. f , Western blot of proximal interacting proteins extracted with streptavidin in hMVEC-D cells expressing VE-cadherin-BirA-HA (VE-BioID) that were treated with DMSO, Dll4, or DAPT, immunoblotted for Trio and LAR. All images representative of at least two independent experiments.

    Journal: Nature

    Article Title: A non-canonical Notch complex regulates adherens junctions and vascular barrier function

    doi: 10.1038/nature24998

    Figure Lengend Snippet: Notch1 regulates VE-cadherin interacting proteins to form the Notch1 mechanosensory complex a , Immunoprecipitation of VE-cadherin from scramble and Notch1-KO cells. Co-immunoprecipitation of candidate Notch1-dependent, VE-cadherin effectors was assessed by immunoblotting for VE-PTP, VEGFR2, and LAR (85 kDa P-subunit). b , Immunoprecipitation of the Rac1 GEF Trio from scramble, Notch1-KO, and LAR-KO cells. Immunoblotting for VE-cadherin was used to assess impaired Trio-VE-cadherin co-immunoprecipitation upon depletion of Notch1 or LAR. c , Western blots of VE-cadherin immunoprecipitated from the lysates of lungs from CDH5-Cre(+); NOTCH1 fl/fl and control CDH5-Cre(+); NOTCH1 fl/fl mice and immunoblotted for LAR. d , Western blots of Trio immunoprecipitated from the lysates of lungs from CDH5-Cre(+); NOTCH1 fl/fl and control CDH5-Cre(+); NOTCH1 fl/fl mice and immunoblotted for VE-cadherin. e , Western blot of proximal interacting proteins extracted with streptavidin from hMVEC-D cells expressing BirA-HA (BioID) or VE-cadherin-BirA-HA (VE-BioID) that were treated with DMSO, Dll4, or DAPT, immunoblotted for HA and Notch1 ICD. f , Western blot of proximal interacting proteins extracted with streptavidin in hMVEC-D cells expressing VE-cadherin-BirA-HA (VE-BioID) that were treated with DMSO, Dll4, or DAPT, immunoblotted for Trio and LAR. All images representative of at least two independent experiments.

    Article Snippet: The soluble supernatants were precleared with protein G sepharose beads and then incubated with protein G sepharose beads with VE-cadherin or Trio antibody (Santa Cruz, 1 mg)for 3 hours at 4C, washed three times with lysis buffer and then processed in SDS–PAGE sample buffer.

    Techniques: Immunoprecipitation, Western Blot, Mouse Assay, Expressing

    Fig. 4. Released E-Cad/CTF2 dissociates from PS1 but remains bound to β-catenin. ( A ) Extracts from STS-treated A431 cells were immunoprecipitated with antibodies against PS1 (I-R222), pre-immune serum (PI-R222), β-catenin or desmoglein, and the immunoprecipitates (IPs) obtained were probed on western blots with anti-E-cadherin antibody C36. For reference, cell lysate was also probed; the asterisk shows IgGs. ( B ) A431 cells treated for 6 h with STS were fractionated into membrane, soluble cytosolic and Triton X-100-insoluble (TX100- insoluble) fractions, and the fractions obtained were then probed on western blots with C36 antibody.

    Journal: The EMBO Journal

    Article Title: A presenilin-1/?-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

    doi: 10.1093/emboj/21.8.1948

    Figure Lengend Snippet: Fig. 4. Released E-Cad/CTF2 dissociates from PS1 but remains bound to β-catenin. ( A ) Extracts from STS-treated A431 cells were immunoprecipitated with antibodies against PS1 (I-R222), pre-immune serum (PI-R222), β-catenin or desmoglein, and the immunoprecipitates (IPs) obtained were probed on western blots with anti-E-cadherin antibody C36. For reference, cell lysate was also probed; the asterisk shows IgGs. ( B ) A431 cells treated for 6 h with STS were fractionated into membrane, soluble cytosolic and Triton X-100-insoluble (TX100- insoluble) fractions, and the fractions obtained were then probed on western blots with C36 antibody.

    Article Snippet: Anti-E-cadherin (clone C36), anti-β-catenin and anti-α-catenin monoclonal antibodies were obtained from BD Transduction Laboratories; antibody H108 against E-cadherin ectodomain was obtained from Santa Cruz Biotechnology, Inc.

    Techniques: Immunoprecipitation, Western Blot

    Fig. 2. A PS1-mediated γ-secretase activity cleaves E-cadherin. ( A ), solubilized in RIPA and blotted with anti-E-cadherin C36 antibody. ( B ) A431 cells were pre-incubated for 30 min in the absence (–) or presence (+) of GM6001 (2.5 µM), Z-DEVD-FMK (DEVD, 50 µM), an inactive analogue of L-685,458 (Control, 0.5 µM) or L-685,458 (0.5 µM). Cells were then treated with STS for 6 h to induce apoptosis, and cell extracts were probed with C36 antibody. Middle and lower panels: the filter was exposed to X-ray films for either 5 or 0.5 min. ( C ) Conditioned media (20 µl) from A431 cells cultured in the absence (–) or presence (+) of GM6001 and treated with STS as above were probed on western blots with anti-E-cadherin ectodomain antibody H108. E-Cad/NTF1 indicates the secreted E-cadherin ectodomain. ( D ) Extracts from HEK293 cells stably transfected with either wild-type (WT) PS1 or vector alone were immunoprecipitated and probed with C36 antibody (upper panels). Lower panel: extracts from the above cells were probed with anti-PS1/NTF antibody R222. ( E ) A431 cells were pre-incubated for 30 min in the absence (–) or presence (+) of GM6001 (2.5 µM). Cells were then treated with STS for 6 h and cell extracts were probed on western blots with either H108 (left panel) or C36 (right panel) antibodies.

    Journal: The EMBO Journal

    Article Title: A presenilin-1/?-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

    doi: 10.1093/emboj/21.8.1948

    Figure Lengend Snippet: Fig. 2. A PS1-mediated γ-secretase activity cleaves E-cadherin. ( A ), solubilized in RIPA and blotted with anti-E-cadherin C36 antibody. ( B ) A431 cells were pre-incubated for 30 min in the absence (–) or presence (+) of GM6001 (2.5 µM), Z-DEVD-FMK (DEVD, 50 µM), an inactive analogue of L-685,458 (Control, 0.5 µM) or L-685,458 (0.5 µM). Cells were then treated with STS for 6 h to induce apoptosis, and cell extracts were probed with C36 antibody. Middle and lower panels: the filter was exposed to X-ray films for either 5 or 0.5 min. ( C ) Conditioned media (20 µl) from A431 cells cultured in the absence (–) or presence (+) of GM6001 and treated with STS as above were probed on western blots with anti-E-cadherin ectodomain antibody H108. E-Cad/NTF1 indicates the secreted E-cadherin ectodomain. ( D ) Extracts from HEK293 cells stably transfected with either wild-type (WT) PS1 or vector alone were immunoprecipitated and probed with C36 antibody (upper panels). Lower panel: extracts from the above cells were probed with anti-PS1/NTF antibody R222. ( E ) A431 cells were pre-incubated for 30 min in the absence (–) or presence (+) of GM6001 (2.5 µM). Cells were then treated with STS for 6 h and cell extracts were probed on western blots with either H108 (left panel) or C36 (right panel) antibodies.

    Article Snippet: Anti-E-cadherin (clone C36), anti-β-catenin and anti-α-catenin monoclonal antibodies were obtained from BD Transduction Laboratories; antibody H108 against E-cadherin ectodomain was obtained from Santa Cruz Biotechnology, Inc.

    Techniques: Activity Assay, Incubation, Cell Culture, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation

    Fig. 7. E-cadherin mutation GGG759-761AAA prevents binding to PS1 and inhibits γ-secretase cleavage of E-cadherin and cytosolic release of catenins. ( A ) Right panel: extracts from A431D cells stably transfected with vector, wild-type E-cadherin (WT E-Cad) or E-cadherin mutant GGG759-761AAA (761AAA) were immunoprecipitated with anti-PS1/NTF antibody R222 (PS1 IP) and the IPs were probed with either anti-E-cadherin antibody C36 (upper panel) or anti-PS1/CTF antibody 33B10 (lower panel). The left panel shows relative E-cadherin levels in transfectants. ( B ) A431D cells transfected with either wild-type E-cadherin or E-cadherin mutant 761AAA were incubated in the absence (–) or presence (+) of ionomycin for 45 min, and RIPA extracts were probed on western blots with C36 antibody (upper panel). Cytosolic fractions of the above cultures were probed on western blots with antibodies against E-cadherin (C36, second panel), β-catenin (third panel) or α-catenin (lower panel). ( C ) Schematic representation of the PS1/γ-secretase-mediated disassembly of CAJs. An MMP-mediated proteolytic activity cleaves the extracellular domain of cytoskeletal E-cadherin and releases E-Cad/NTF1 to the extracellular medium (a). Fragment E-Cad/CTF1 containing the transmembrane and cytoplasmic sequence of E-cadherin remains bound to PS1, β-catenin, α-catenin and the actin cytoskeleton. E-Cad/CTF1 is then cleaved by a PS1/γ-secretase activity at the membrane–cytosol interface to produce E-Cad/CTF2, which dissociates from both PS1 and F-actin and is released to the cytosol in a complex with β-catenin (b). Full-length E-cadherin bound to the cytoskeleton can also be cleaved by the PS1/γ-secretase activity (c). No E-Cad/CTF2–α-catenin complex was detected, suggesting that α-catenin dissociates from E-Cad/CTF2 (unpublished observations). α, α-catenin; β, β-catenin.

    Journal: The EMBO Journal

    Article Title: A presenilin-1/?-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

    doi: 10.1093/emboj/21.8.1948

    Figure Lengend Snippet: Fig. 7. E-cadherin mutation GGG759-761AAA prevents binding to PS1 and inhibits γ-secretase cleavage of E-cadherin and cytosolic release of catenins. ( A ) Right panel: extracts from A431D cells stably transfected with vector, wild-type E-cadherin (WT E-Cad) or E-cadherin mutant GGG759-761AAA (761AAA) were immunoprecipitated with anti-PS1/NTF antibody R222 (PS1 IP) and the IPs were probed with either anti-E-cadherin antibody C36 (upper panel) or anti-PS1/CTF antibody 33B10 (lower panel). The left panel shows relative E-cadherin levels in transfectants. ( B ) A431D cells transfected with either wild-type E-cadherin or E-cadherin mutant 761AAA were incubated in the absence (–) or presence (+) of ionomycin for 45 min, and RIPA extracts were probed on western blots with C36 antibody (upper panel). Cytosolic fractions of the above cultures were probed on western blots with antibodies against E-cadherin (C36, second panel), β-catenin (third panel) or α-catenin (lower panel). ( C ) Schematic representation of the PS1/γ-secretase-mediated disassembly of CAJs. An MMP-mediated proteolytic activity cleaves the extracellular domain of cytoskeletal E-cadherin and releases E-Cad/NTF1 to the extracellular medium (a). Fragment E-Cad/CTF1 containing the transmembrane and cytoplasmic sequence of E-cadherin remains bound to PS1, β-catenin, α-catenin and the actin cytoskeleton. E-Cad/CTF1 is then cleaved by a PS1/γ-secretase activity at the membrane–cytosol interface to produce E-Cad/CTF2, which dissociates from both PS1 and F-actin and is released to the cytosol in a complex with β-catenin (b). Full-length E-cadherin bound to the cytoskeleton can also be cleaved by the PS1/γ-secretase activity (c). No E-Cad/CTF2–α-catenin complex was detected, suggesting that α-catenin dissociates from E-Cad/CTF2 (unpublished observations). α, α-catenin; β, β-catenin.

    Article Snippet: Anti-E-cadherin (clone C36), anti-β-catenin and anti-α-catenin monoclonal antibodies were obtained from BD Transduction Laboratories; antibody H108 against E-cadherin ectodomain was obtained from Santa Cruz Biotechnology, Inc.

    Techniques: Mutagenesis, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Western Blot, Activity Assay, Sequencing

    Fig. 1. A PS1-mediated γ-secretase activity controls E-cadherin processing. ( A ) Extracts from PS1+/+ or PS1–/– mouse embryos were probed on western blots with either anti-cytoplasmic E-cadherin C36 (upper panel) or anti-cytoplasmic APP R1 (middle panels) antibodies. E-Cad/FL denotes full-length E-cadherin. The asterisk identifies mouse IgGs. Lower panel: extract probed with anti-PS1/CTF antibody 33B10. ( B ) Extracts from E-cadherin-transfected PS1+/+ or PS1–/– mouse fibroblasts were probed with anti-E-cadherin C36 (upper panel) or 33B10 (lower panel) antibodies. ( C ) PS1+/+ fibroblasts were treated for 6 h either with the γ-secretase inhibitor L-685,458 (0.5 µM) or with dimethylsulfoxide. Extracts from these cell cultures were then probed with anti-E-cadherin C36. The asterisk indicates a non-specific band.

    Journal: The EMBO Journal

    Article Title: A presenilin-1/?-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

    doi: 10.1093/emboj/21.8.1948

    Figure Lengend Snippet: Fig. 1. A PS1-mediated γ-secretase activity controls E-cadherin processing. ( A ) Extracts from PS1+/+ or PS1–/– mouse embryos were probed on western blots with either anti-cytoplasmic E-cadherin C36 (upper panel) or anti-cytoplasmic APP R1 (middle panels) antibodies. E-Cad/FL denotes full-length E-cadherin. The asterisk identifies mouse IgGs. Lower panel: extract probed with anti-PS1/CTF antibody 33B10. ( B ) Extracts from E-cadherin-transfected PS1+/+ or PS1–/– mouse fibroblasts were probed with anti-E-cadherin C36 (upper panel) or 33B10 (lower panel) antibodies. ( C ) PS1+/+ fibroblasts were treated for 6 h either with the γ-secretase inhibitor L-685,458 (0.5 µM) or with dimethylsulfoxide. Extracts from these cell cultures were then probed with anti-E-cadherin C36. The asterisk indicates a non-specific band.

    Article Snippet: Anti-E-cadherin (clone C36), anti-β-catenin and anti-α-catenin monoclonal antibodies were obtained from BD Transduction Laboratories; antibody H108 against E-cadherin ectodomain was obtained from Santa Cruz Biotechnology, Inc.

    Techniques: Activity Assay, Western Blot, Transfection

    H. pylori infection of primary-cell preparations and NCI-N87 cells has no effect on IQGAP-1 or E-cadherin protein expression. Ten microliters of H. pylori with an OD 600 of 0.5 was used to inoculate 1 ml of growth medium. The infected epithelial cells were maintained in 10% CO 2 at 37°C for 48 h prior to collection of protein. Whole-cell lysates were prepared from control and infected-cell preparations, and protein was analyzed by SDS-PAGE using 7.5% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were incubated with monoclonal anti-IQGAP-1 and monoclonal anti-E-cadherin antibodies. The density of the bands was related to that of the housekeeping protein histone 2B. Data are representative of three to six independent experiments.

    Journal: Infection and Immunity

    Article Title: Helicobacter pylori Infection Targets Adherens Junction Regulatory Proteins and Results in Increased Rates of Migration in Human Gastric Epithelial Cells

    doi: 10.1128/IAI.72.9.5181-5192.2004

    Figure Lengend Snippet: H. pylori infection of primary-cell preparations and NCI-N87 cells has no effect on IQGAP-1 or E-cadherin protein expression. Ten microliters of H. pylori with an OD 600 of 0.5 was used to inoculate 1 ml of growth medium. The infected epithelial cells were maintained in 10% CO 2 at 37°C for 48 h prior to collection of protein. Whole-cell lysates were prepared from control and infected-cell preparations, and protein was analyzed by SDS-PAGE using 7.5% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were incubated with monoclonal anti-IQGAP-1 and monoclonal anti-E-cadherin antibodies. The density of the bands was related to that of the housekeeping protein histone 2B. Data are representative of three to six independent experiments.

    Article Snippet: The cells were lysed with lysis buffer (50 mM Tris [pH 7.5], 1% Triton X-100, 500 μl of 1× complete protease inhibitors [Roche]), and the lysates were incubated for 1 to 2 h at 4°C with or without mouse anti-E-cadherin (BD Transduction Laboratories), followed by a 0.5-h incubation with protein G beads (Amersham Pharmacia).

    Techniques: Infection, Expressing, SDS Page, Incubation

    Primary-cell preparations immunostained for IQGAP-1 and E-cadherin. Primary-cell preparations were plated on APES-coated glass coverslips and incubated for 48 h to facilitate attachment prior to infection with H. pylori . Control and infected cells were fixed 48 h postinfection in 4% paraformaldehyde for 10 min at room temperature. (A) Control (Cnt) cells were immunostained with monoclonal anti-IQGAP-1 antibodies for 18 h at 4°C. Note the presence of IR at adherens junctions (arrow). (B) Control cells were immunostained with monoclonal anti-E-cadherin antibodies for 18 h at 4°C. (C) Cells were infected with H. pylori and immunostained for IQGAP-1. Note the dramatic increase in internalized protein in tubulovesicles (arrows). (D) Cells were infected with H. pylori and immunostained for E-cadherin. Note the presence of internalized protein in small vesicles. Scale bar = 5 μm. Images are representative of three independent experiments.

    Journal: Infection and Immunity

    Article Title: Helicobacter pylori Infection Targets Adherens Junction Regulatory Proteins and Results in Increased Rates of Migration in Human Gastric Epithelial Cells

    doi: 10.1128/IAI.72.9.5181-5192.2004

    Figure Lengend Snippet: Primary-cell preparations immunostained for IQGAP-1 and E-cadherin. Primary-cell preparations were plated on APES-coated glass coverslips and incubated for 48 h to facilitate attachment prior to infection with H. pylori . Control and infected cells were fixed 48 h postinfection in 4% paraformaldehyde for 10 min at room temperature. (A) Control (Cnt) cells were immunostained with monoclonal anti-IQGAP-1 antibodies for 18 h at 4°C. Note the presence of IR at adherens junctions (arrow). (B) Control cells were immunostained with monoclonal anti-E-cadherin antibodies for 18 h at 4°C. (C) Cells were infected with H. pylori and immunostained for IQGAP-1. Note the dramatic increase in internalized protein in tubulovesicles (arrows). (D) Cells were infected with H. pylori and immunostained for E-cadherin. Note the presence of internalized protein in small vesicles. Scale bar = 5 μm. Images are representative of three independent experiments.

    Article Snippet: The cells were lysed with lysis buffer (50 mM Tris [pH 7.5], 1% Triton X-100, 500 μl of 1× complete protease inhibitors [Roche]), and the lysates were incubated for 1 to 2 h at 4°C with or without mouse anti-E-cadherin (BD Transduction Laboratories), followed by a 0.5-h incubation with protein G beads (Amersham Pharmacia).

    Techniques: Incubation, Infection

    Immunocytochemistry shows that the primary and NCI-N87 cells show similar patterns of E-cadherin and actin staining. All three cell preparations were grown on APES-coated coverslips for 48 h prior to fixation with 4% paraformaldehyde and permeabilization with 0.1% Triton X-100. All cells were immunostained with a monoclonal anti-E-cadherin antibody for 18 h at 4°C before being incubated with phalloidin-AlexaFluor 594 for 1 h at room temperature. At the basolateral membrane E-cadherin (A, primary; E, NCI-N87) is reduced and actin stress fibers predominate (B, primary; F, NCI-N87). From 1 μm from the substrate to the top of the cells both showed strong E-cadherin (C, primary; G, NCI-N87) and cortical actin (D, primary; H, NCI-N87) staining at the plasma membrane. AGS cells show a punctate pattern of E-cadherin staining that does not localize to adherens junctions (I). AGS cells have a uniform pattern of filamentous actin staining with a small number of stress fibers (arrows) and cortical actin (arrowheads) (J). Scale bar = 10 μm. Images are representative of three individual experiments.

    Journal: Infection and Immunity

    Article Title: Helicobacter pylori Infection Targets Adherens Junction Regulatory Proteins and Results in Increased Rates of Migration in Human Gastric Epithelial Cells

    doi: 10.1128/IAI.72.9.5181-5192.2004

    Figure Lengend Snippet: Immunocytochemistry shows that the primary and NCI-N87 cells show similar patterns of E-cadherin and actin staining. All three cell preparations were grown on APES-coated coverslips for 48 h prior to fixation with 4% paraformaldehyde and permeabilization with 0.1% Triton X-100. All cells were immunostained with a monoclonal anti-E-cadherin antibody for 18 h at 4°C before being incubated with phalloidin-AlexaFluor 594 for 1 h at room temperature. At the basolateral membrane E-cadherin (A, primary; E, NCI-N87) is reduced and actin stress fibers predominate (B, primary; F, NCI-N87). From 1 μm from the substrate to the top of the cells both showed strong E-cadherin (C, primary; G, NCI-N87) and cortical actin (D, primary; H, NCI-N87) staining at the plasma membrane. AGS cells show a punctate pattern of E-cadherin staining that does not localize to adherens junctions (I). AGS cells have a uniform pattern of filamentous actin staining with a small number of stress fibers (arrows) and cortical actin (arrowheads) (J). Scale bar = 10 μm. Images are representative of three individual experiments.

    Article Snippet: The cells were lysed with lysis buffer (50 mM Tris [pH 7.5], 1% Triton X-100, 500 μl of 1× complete protease inhibitors [Roche]), and the lysates were incubated for 1 to 2 h at 4°C with or without mouse anti-E-cadherin (BD Transduction Laboratories), followed by a 0.5-h incubation with protein G beads (Amersham Pharmacia).

    Techniques: Immunocytochemistry, Staining, Incubation

    Enzymatic activity of cathepsin G is required for cathepsin G-promoted E-cadherin-mediated cell-cell adhesion. MCF-7 cells were incubated in 5% FBS-containing medium on fibronectin for 24 hours. (a) After washing, the adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 24 hours. Cathepsin G-induced cell condensation was observed by phase-contrast microscopy. (b) After washing, adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 3 hours. The immunocomplexes with anti-E-cadherin were then analyzed by immunoblotting with anti-E-cadherin using an anti- β -catenin antibody.

    Journal: Mediators of Inflammation

    Article Title: Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    doi: 10.1155/2009/850940

    Figure Lengend Snippet: Enzymatic activity of cathepsin G is required for cathepsin G-promoted E-cadherin-mediated cell-cell adhesion. MCF-7 cells were incubated in 5% FBS-containing medium on fibronectin for 24 hours. (a) After washing, the adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 24 hours. Cathepsin G-induced cell condensation was observed by phase-contrast microscopy. (b) After washing, adherent cells were treated with cathepsin G in the absence or presence of 50 μ g/mL chymostatin or 200 μ g/mL α 1 -antitrypsin (AT) for 3 hours. The immunocomplexes with anti-E-cadherin were then analyzed by immunoblotting with anti-E-cadherin using an anti- β -catenin antibody.

    Article Snippet: The immunological reagents used were anti-α -catenin (1G5), anti-β -catenin (E-5), anti-E-cadherin (67A4 and G-10), anti-Rap1 (5G7), anti-PKCμ /PKD1(C-20), and HRP-conjugated antimouse IgG1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activity Assay, Incubation, Microscopy

    Cathepsin G-induced E-cadherin/catenin complex formation and E-cadherin-mediated cell-cell adhesion of MCF-7 cells. MCF-7 cells were cultured in dishes coated with fibronectin for 24 hours. After washing, adherent cells were incubated in serum-free medium without or with 0.5 mU/mL cathepsin G. At each indicated culture time, the cells were lysed, and E-cadherin/catenin complex formation of MCF-7 cells was analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) and (b) Immunocomplexes with anti-E-cadherin were analyzed by immunoblotting using an anti- β -catenin (a) or anti- α -catenin antibody (b). Whole-cell lysates (WCLs) were immunoblotted with an anti- β -catenin (a) or anti- α -catenin antibody (b). (c) BALB-MC.E12 mouse mammary tumor cells were analyzed as shown in (a). (d) Treatments inhibiting E-cadherin-mediated cell-cell adhesion disrupt cathepsin G-induced cell condensation. MCF-7 cells were cultured in 5% FBS-containing medium on fibronectin for 24 hours. After washing, cell condensation was induced by cathepsin G for 24 hours. Condensed cells were then subjected to serum-free medium supplemented with 400 μ M EGTA for 6 hours or HECD-1 (100 μ g/mL) for 24 hours and then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200.

    Journal: Mediators of Inflammation

    Article Title: Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    doi: 10.1155/2009/850940

    Figure Lengend Snippet: Cathepsin G-induced E-cadherin/catenin complex formation and E-cadherin-mediated cell-cell adhesion of MCF-7 cells. MCF-7 cells were cultured in dishes coated with fibronectin for 24 hours. After washing, adherent cells were incubated in serum-free medium without or with 0.5 mU/mL cathepsin G. At each indicated culture time, the cells were lysed, and E-cadherin/catenin complex formation of MCF-7 cells was analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) and (b) Immunocomplexes with anti-E-cadherin were analyzed by immunoblotting using an anti- β -catenin (a) or anti- α -catenin antibody (b). Whole-cell lysates (WCLs) were immunoblotted with an anti- β -catenin (a) or anti- α -catenin antibody (b). (c) BALB-MC.E12 mouse mammary tumor cells were analyzed as shown in (a). (d) Treatments inhibiting E-cadherin-mediated cell-cell adhesion disrupt cathepsin G-induced cell condensation. MCF-7 cells were cultured in 5% FBS-containing medium on fibronectin for 24 hours. After washing, cell condensation was induced by cathepsin G for 24 hours. Condensed cells were then subjected to serum-free medium supplemented with 400 μ M EGTA for 6 hours or HECD-1 (100 μ g/mL) for 24 hours and then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200.

    Article Snippet: The immunological reagents used were anti-α -catenin (1G5), anti-β -catenin (E-5), anti-E-cadherin (67A4 and G-10), anti-Rap1 (5G7), anti-PKCμ /PKD1(C-20), and HRP-conjugated antimouse IgG1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Culture, Incubation, Immunoprecipitation, Western Blot, Microscopy

    Cathepsin G promotes E-cadherin/PKD1 complex formation in MCF-7 cells. MCF-7 cells treated with cathepsin G for 10 hours were analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) Immunocomplexes with anti-PKD1 antibody or whole-cell lysates (WCLs) were analyzed by immunoblotting using an anti-E-cadherin antibody. (b) After washing, the adherent cells were treated with cathepsin G in the absence or presence of 5 μ M Go6976 or 5 μ M of the negative control compound bisindolylmaleimide V for 5 hours. Cells were then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200. Arrowheads indicate the site of cell condensation in the cathepsin G-treated cells, while cell condensation was not observed in the cells treated with cathepsin G and Go6976.

    Journal: Mediators of Inflammation

    Article Title: Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    doi: 10.1155/2009/850940

    Figure Lengend Snippet: Cathepsin G promotes E-cadherin/PKD1 complex formation in MCF-7 cells. MCF-7 cells treated with cathepsin G for 10 hours were analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) Immunocomplexes with anti-PKD1 antibody or whole-cell lysates (WCLs) were analyzed by immunoblotting using an anti-E-cadherin antibody. (b) After washing, the adherent cells were treated with cathepsin G in the absence or presence of 5 μ M Go6976 or 5 μ M of the negative control compound bisindolylmaleimide V for 5 hours. Cells were then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200. Arrowheads indicate the site of cell condensation in the cathepsin G-treated cells, while cell condensation was not observed in the cells treated with cathepsin G and Go6976.

    Article Snippet: The immunological reagents used were anti-α -catenin (1G5), anti-β -catenin (E-5), anti-E-cadherin (67A4 and G-10), anti-Rap1 (5G7), anti-PKCμ /PKD1(C-20), and HRP-conjugated antimouse IgG1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Immunoprecipitation, Western Blot, Negative Control, Microscopy

    Expression of Smad3 linker mutant that lacks phosphorylation sites attenuates TGF-β1–induced EMT, migration, and MMP2 activation in A549 lung cancer cells. (A) Schematic representation of Smad3 wild type (S3 WT ) and mutant lacking phosphorylation sites in the linker region (S3 EPSM ). A549 cells infected with lentivirus carrying expression vector for Myc-tagged Smad3-WT (Myc-S3 WT ) or Myc-tagged Smad3-EPSM (Myc-S3 EPSM ), or its corresponding empty vector (pCAG) were stimulated with 5 ng/ml of TGF-β1 for 36 hours and then subjected to Western blot analysis using anti-E-cadherin, anti-N-cadherin, anti-vimentin, and anti-Myc antibodies (B) and immunofluorescence staining for E-cadherin and Myc-S3 EPSM (C). (D) A549 cells (1 × 10 4 ) that were infected as in B were seeded on 8-mm porous Transwell chambers and then stimulated with treatment with 5 ng/ml of TGF-β1 for 36 hours. Transmigrating cells were stained and counted as in Figure 2 B . Quantitative data are shown as the mean ± SD of three independent experiments. ** P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179

    doi: 10.1016/j.neo.2015.06.004

    Figure Lengend Snippet: Expression of Smad3 linker mutant that lacks phosphorylation sites attenuates TGF-β1–induced EMT, migration, and MMP2 activation in A549 lung cancer cells. (A) Schematic representation of Smad3 wild type (S3 WT ) and mutant lacking phosphorylation sites in the linker region (S3 EPSM ). A549 cells infected with lentivirus carrying expression vector for Myc-tagged Smad3-WT (Myc-S3 WT ) or Myc-tagged Smad3-EPSM (Myc-S3 EPSM ), or its corresponding empty vector (pCAG) were stimulated with 5 ng/ml of TGF-β1 for 36 hours and then subjected to Western blot analysis using anti-E-cadherin, anti-N-cadherin, anti-vimentin, and anti-Myc antibodies (B) and immunofluorescence staining for E-cadherin and Myc-S3 EPSM (C). (D) A549 cells (1 × 10 4 ) that were infected as in B were seeded on 8-mm porous Transwell chambers and then stimulated with treatment with 5 ng/ml of TGF-β1 for 36 hours. Transmigrating cells were stained and counted as in Figure 2 B . Quantitative data are shown as the mean ± SD of three independent experiments. ** P

    Article Snippet: The membranes were subsequently blocked for 1 hour with Tris-buffered saline containing 0.05% (v/v) Tween 20 and 5% (w/v) nonfat dry milk and then incubated with appropriate antibodies [anti-myc and anti-vimentin (Santa Cruz Biotechnology), anti-Smad2 and anti-Smad3 (Zymed), anti-phospho-Smad3 (Ser423/425) and anti-phospho-Akt (Ser426) (Cell Signaling Technology, Danvers, MA), and anti-E-cadherin and anti–N-cadherin (BD Transduction laboratories, Lexington, KY), anti–smooth muscle α-actin and anti–β-actin (Sigma-Aldrich)].

    Techniques: Expressing, Mutagenesis, Migration, Activation Assay, Infection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining

    PI3K/Akt1 pathway is crucial for TGF-β1–induced EMT, cell migration, and MMP2 activation and is inhibited by KF. (A) A549 cells were treated as in Figure 4 A except for the TGF-β1 treatment for 90 minutes. (B) A549 cells were pretreated with DMSO or AktIV at the indicated concentrations for 30 minutes and then stimulated with 5 ng/ml of TGF-β1 for 48 hours. All cells were then subjected to Western blot analysis using anti–phospho-Akt, anti–E-cadherin, and anti–N-cadherin antibodies. β-Actin levels were monitored as a loading control for whole extracts. (C) A549 cells were transiently transfected with E-cadherin promoter-reporter (E-cadherin-Luc) combination with constitutively active TβRI (CA-TβRI, TβRI-T204D)-, dominant negative p85 (DN-p85, p85ΔiSH2-N)-, and dominant negative Akt1 (DN-Akt1, Akt1-K179M)-expressing plasmids and the corresponding empty vector (pcDNA). All quantitative data are the mean ± SD of three independent experiments. ** P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179

    doi: 10.1016/j.neo.2015.06.004

    Figure Lengend Snippet: PI3K/Akt1 pathway is crucial for TGF-β1–induced EMT, cell migration, and MMP2 activation and is inhibited by KF. (A) A549 cells were treated as in Figure 4 A except for the TGF-β1 treatment for 90 minutes. (B) A549 cells were pretreated with DMSO or AktIV at the indicated concentrations for 30 minutes and then stimulated with 5 ng/ml of TGF-β1 for 48 hours. All cells were then subjected to Western blot analysis using anti–phospho-Akt, anti–E-cadherin, and anti–N-cadherin antibodies. β-Actin levels were monitored as a loading control for whole extracts. (C) A549 cells were transiently transfected with E-cadherin promoter-reporter (E-cadherin-Luc) combination with constitutively active TβRI (CA-TβRI, TβRI-T204D)-, dominant negative p85 (DN-p85, p85ΔiSH2-N)-, and dominant negative Akt1 (DN-Akt1, Akt1-K179M)-expressing plasmids and the corresponding empty vector (pcDNA). All quantitative data are the mean ± SD of three independent experiments. ** P

    Article Snippet: The membranes were subsequently blocked for 1 hour with Tris-buffered saline containing 0.05% (v/v) Tween 20 and 5% (w/v) nonfat dry milk and then incubated with appropriate antibodies [anti-myc and anti-vimentin (Santa Cruz Biotechnology), anti-Smad2 and anti-Smad3 (Zymed), anti-phospho-Smad3 (Ser423/425) and anti-phospho-Akt (Ser426) (Cell Signaling Technology, Danvers, MA), and anti-E-cadherin and anti–N-cadherin (BD Transduction laboratories, Lexington, KY), anti–smooth muscle α-actin and anti–β-actin (Sigma-Aldrich)].

    Techniques: Migration, Activation Assay, Western Blot, Transfection, Dominant Negative Mutation, Expressing, Plasmid Preparation

    Stabilization of endothelial VE-cadherin junctions blocks melanoma-induced gap formation and subsequent endothelial barrier breakdown. HPMEC monolayers were treated with FGF1 for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. Results represent the mean +/−SEM, (***p

    Journal: Scientific Reports

    Article Title: VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

    doi: 10.1038/srep45835

    Figure Lengend Snippet: Stabilization of endothelial VE-cadherin junctions blocks melanoma-induced gap formation and subsequent endothelial barrier breakdown. HPMEC monolayers were treated with FGF1 for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. Results represent the mean +/−SEM, (***p

    Article Snippet: Cells were then incubated with a primary antibody for VE-cadherin (1:300 dilution; Cell Signaling), in an antibody dilution buffer (ADB) containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS over night at 4 °C.

    Techniques: Cell Culture

    Endothelial cell contractility is necessary for melanoma-induced gap formation and subsequent endothelial barrier breakdown. ( A ) HPMEC monolayers stimulated with thrombin or cultured in direct contact with either A2058 (metastatic) or WM35 (non-metastatic) melanoma cells. Endothelial cell contractility was assessed via immunostaining of F-Actin (red) and ppMLC (green). Anisotropy numbers on F-actin images represent a measurement of fiber alignment (mean +/−SEM, n = 3). Scale bars: 10 μm. ( B ) Phosphorylation levels of MLC were determined by measuring mean fluorescence intensity of ppMLC images. Results represent the mean +/−SEM (n = 3). ( C ) Co-localization of ppMLC and F-actin filaments was measured from immunofluorescence images and is reported using Pearson’s correlation coefficient. Results represent the mean +/−SEM (n = 3). ( D ) HPMEC monolayers were pre-treated with inhibitors of contractility for 30 min and were then co-cultured in direct contact with A2058 melanoma cells for 45 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within the gap regions over the number of pixels in the entire image. Results represent the mean ± SEM. A significant difference was found for the A2058 group between no treatment or DMSO and all the inhibitors. Also, significant differences were found between control and A2058 for no treatment and DMSO groups. No significant differences were observed between control and A2058 treated monolayers for any of the inhibitor treatments. ( E ) HPMEC monolayers cultured on 8 μm polycarbonate membranes were pre-treated with blebbistatin for 30 min and WM35 or A2058 melanoma cell migration across the monolayer was assessed. Results represent mean +/− SEM. (***p

    Journal: Scientific Reports

    Article Title: VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

    doi: 10.1038/srep45835

    Figure Lengend Snippet: Endothelial cell contractility is necessary for melanoma-induced gap formation and subsequent endothelial barrier breakdown. ( A ) HPMEC monolayers stimulated with thrombin or cultured in direct contact with either A2058 (metastatic) or WM35 (non-metastatic) melanoma cells. Endothelial cell contractility was assessed via immunostaining of F-Actin (red) and ppMLC (green). Anisotropy numbers on F-actin images represent a measurement of fiber alignment (mean +/−SEM, n = 3). Scale bars: 10 μm. ( B ) Phosphorylation levels of MLC were determined by measuring mean fluorescence intensity of ppMLC images. Results represent the mean +/−SEM (n = 3). ( C ) Co-localization of ppMLC and F-actin filaments was measured from immunofluorescence images and is reported using Pearson’s correlation coefficient. Results represent the mean +/−SEM (n = 3). ( D ) HPMEC monolayers were pre-treated with inhibitors of contractility for 30 min and were then co-cultured in direct contact with A2058 melanoma cells for 45 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within the gap regions over the number of pixels in the entire image. Results represent the mean ± SEM. A significant difference was found for the A2058 group between no treatment or DMSO and all the inhibitors. Also, significant differences were found between control and A2058 for no treatment and DMSO groups. No significant differences were observed between control and A2058 treated monolayers for any of the inhibitor treatments. ( E ) HPMEC monolayers cultured on 8 μm polycarbonate membranes were pre-treated with blebbistatin for 30 min and WM35 or A2058 melanoma cell migration across the monolayer was assessed. Results represent mean +/− SEM. (***p

    Article Snippet: Cells were then incubated with a primary antibody for VE-cadherin (1:300 dilution; Cell Signaling), in an antibody dilution buffer (ADB) containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS over night at 4 °C.

    Techniques: Cell Culture, Immunostaining, Fluorescence, Immunofluorescence, Migration

    Metastatic melanoma cells use IL-8 signalling and VLA-4/VCAM-1 interactions to induce gap formation and subsequent endothelial barrier breakdown. ( A ) HPMEC monolayers were pre-treated with neutralizing antibodies against CXCR1 and CXCR2 receptors for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. ( B ) HPMEC monolayers were cultured in direct contact with either A2058 melanoma cells alone or A2058 melanoma cells pre-treated with VLA-4 neutralizing antibodies. Results represent the mean +/−SEM, (***p

    Journal: Scientific Reports

    Article Title: VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

    doi: 10.1038/srep45835

    Figure Lengend Snippet: Metastatic melanoma cells use IL-8 signalling and VLA-4/VCAM-1 interactions to induce gap formation and subsequent endothelial barrier breakdown. ( A ) HPMEC monolayers were pre-treated with neutralizing antibodies against CXCR1 and CXCR2 receptors for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. ( B ) HPMEC monolayers were cultured in direct contact with either A2058 melanoma cells alone or A2058 melanoma cells pre-treated with VLA-4 neutralizing antibodies. Results represent the mean +/−SEM, (***p

    Article Snippet: Cells were then incubated with a primary antibody for VE-cadherin (1:300 dilution; Cell Signaling), in an antibody dilution buffer (ADB) containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS over night at 4 °C.

    Techniques: Cell Culture

    Metastatic melanoma cells induce VE-cadherin phosphorylation via Src activation. ( A ) Western blot and densitometric analysis of total and phosphorylated VE-cadherin in endothelial cells in co-culture with either A2058 or WM35 melanoma cells. Endothelial cells were either left untreated or pre-treated with the Src inhibitor PP1 prior to co-culture with melanoma cells. ( B ) Time-lapse images of endothelial cells expressing the Src FRET biosensor stimulated with either A2058 metastatic melanoma cells WM35 non-metastatic melanoma cells, or media only. Images are pseudocolored showing CFP/FRET ratio. Scale bars represent 5 μm. ( C ) Quantification of CFP/FRET ratio normalized to background signal (before stimulation) for each time-lapse image set and then normalized to negative control. Plot represent mean +/−SEM (n = 6).

    Journal: Scientific Reports

    Article Title: VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

    doi: 10.1038/srep45835

    Figure Lengend Snippet: Metastatic melanoma cells induce VE-cadherin phosphorylation via Src activation. ( A ) Western blot and densitometric analysis of total and phosphorylated VE-cadherin in endothelial cells in co-culture with either A2058 or WM35 melanoma cells. Endothelial cells were either left untreated or pre-treated with the Src inhibitor PP1 prior to co-culture with melanoma cells. ( B ) Time-lapse images of endothelial cells expressing the Src FRET biosensor stimulated with either A2058 metastatic melanoma cells WM35 non-metastatic melanoma cells, or media only. Images are pseudocolored showing CFP/FRET ratio. Scale bars represent 5 μm. ( C ) Quantification of CFP/FRET ratio normalized to background signal (before stimulation) for each time-lapse image set and then normalized to negative control. Plot represent mean +/−SEM (n = 6).

    Article Snippet: Cells were then incubated with a primary antibody for VE-cadherin (1:300 dilution; Cell Signaling), in an antibody dilution buffer (ADB) containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS over night at 4 °C.

    Techniques: Activation Assay, Western Blot, Co-Culture Assay, Expressing, Negative Control

    Metastatic melanoma cells induce gap formation between endothelial cells. ( A ) HPMEC monolayers cultured in direct contact with either A2058 (metastatic) or WM35 (non-metastatic) melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin (green). Yellow outlines show intercellular gaps, arrows show adherens junctions and arrowheads show either A2058 or WM35 melanoma cells. Scale bars: 50 μm. ( B ) The percentage of endothelial gaps was quantified as the number of pixels within the gap regions divided by the number of pixels in the entire image. Results represent the mean +/−SEM (***p

    Journal: Scientific Reports

    Article Title: VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

    doi: 10.1038/srep45835

    Figure Lengend Snippet: Metastatic melanoma cells induce gap formation between endothelial cells. ( A ) HPMEC monolayers cultured in direct contact with either A2058 (metastatic) or WM35 (non-metastatic) melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin (green). Yellow outlines show intercellular gaps, arrows show adherens junctions and arrowheads show either A2058 or WM35 melanoma cells. Scale bars: 50 μm. ( B ) The percentage of endothelial gaps was quantified as the number of pixels within the gap regions divided by the number of pixels in the entire image. Results represent the mean +/−SEM (***p

    Article Snippet: Cells were then incubated with a primary antibody for VE-cadherin (1:300 dilution; Cell Signaling), in an antibody dilution buffer (ADB) containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS over night at 4 °C.

    Techniques: Cell Culture

    Soluble and receptor-ligand interactions activate Src in endothelial cells resulting in VE-cadherin phosphorylation. ( A ) Western blot and densitometric analysis of total and phosphorylated VE-cadherin induced by IL-8 [100, 20 or 10 ng/ml] after 0, 10, 45 and 90 min. ( B ) Western blot and densitometric analysis of total and phosphorylated VE-cadherin induced by K562-WT or K562-VLA-4 cells after 0, 10, 45 and 90 min. ( C ) Time-lapse images of endothelial cells expressing the Src FRET biosensor stimulated with different concentrations of IL-8 [100, 20 or 10 ng/ml] or with anti-VCAM-1 antibody [30 μg/ml]. Images are pseudocolored showing CFP/FRET ratio (Bars represent 5 μm). ( D,E ) Quantification of CFP/FRET ratio normalized to background signal (before stimulation) for each time-lapse image set and then normalized to negative control. Plots represent mean +/−SEM (n = 6).

    Journal: Scientific Reports

    Article Title: VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

    doi: 10.1038/srep45835

    Figure Lengend Snippet: Soluble and receptor-ligand interactions activate Src in endothelial cells resulting in VE-cadherin phosphorylation. ( A ) Western blot and densitometric analysis of total and phosphorylated VE-cadherin induced by IL-8 [100, 20 or 10 ng/ml] after 0, 10, 45 and 90 min. ( B ) Western blot and densitometric analysis of total and phosphorylated VE-cadherin induced by K562-WT or K562-VLA-4 cells after 0, 10, 45 and 90 min. ( C ) Time-lapse images of endothelial cells expressing the Src FRET biosensor stimulated with different concentrations of IL-8 [100, 20 or 10 ng/ml] or with anti-VCAM-1 antibody [30 μg/ml]. Images are pseudocolored showing CFP/FRET ratio (Bars represent 5 μm). ( D,E ) Quantification of CFP/FRET ratio normalized to background signal (before stimulation) for each time-lapse image set and then normalized to negative control. Plots represent mean +/−SEM (n = 6).

    Article Snippet: Cells were then incubated with a primary antibody for VE-cadherin (1:300 dilution; Cell Signaling), in an antibody dilution buffer (ADB) containing 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS over night at 4 °C.

    Techniques: Western Blot, Expressing, Negative Control

    Ngn1 mRNA expression decreases with age; N-cadherin regulates Ngn1 mRNA expression. Panel A shows Ngn1 mRNA expression measured by RT-PCR in young adult and retired breeder neurospheres treated with or without DETA-NONOate for 7 days. Panels B and C show quantitative data of Ngn1 mRNA expression in young neurospheres (B) and retired breeder (C) neurosphere treated with or without DETA-NONOate, N-cadherin, anti-N-cadherin and DETA-NONOate combination with anti-N-cadherin.

    Journal: Neuroscience

    Article Title: N-CADHERIN MEDIATES NITRIC OXIDE-INDUCED NEUROGENESIS IN YOUNG AND RETIRED BREEDER NEUROSPHERES

    doi: 10.1016/j.neuroscience.2006.02.064

    Figure Lengend Snippet: Ngn1 mRNA expression decreases with age; N-cadherin regulates Ngn1 mRNA expression. Panel A shows Ngn1 mRNA expression measured by RT-PCR in young adult and retired breeder neurospheres treated with or without DETA-NONOate for 7 days. Panels B and C show quantitative data of Ngn1 mRNA expression in young neurospheres (B) and retired breeder (C) neurosphere treated with or without DETA-NONOate, N-cadherin, anti-N-cadherin and DETA-NONOate combination with anti-N-cadherin.

    Article Snippet: To further investigate whether N-cadherin participates in DETA-NONOate-induced neuronal differentiation, N-cadherin (5 μ g/cm2 ) (recombinant human N-cadherin/Fc chimera, R & D System) and neutralized anti-N-cadherin antibody (monoclonal anti-N-cadherin/A-CAM; Sigma, St. Louis, MO, USA) treatment was employed.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    N-cadherin regulates neurosphere formation and cell adhesion. Panel A shows quantitative data of N-cadherin mRNA expression in young and retired breeder rat SVZ neurospheres treated with or without DETA-NONOate for 7 days. Panels B–F show young adult neurosphere formation in control (B), treated with 0.4 μ M DETANONOate (C), treated with N-cadherin (5 μ g/cm 2 ) alone (D), treated with anti-N-cadherin antibody (80 ng/ml) alone (E), treated with anti-N-cadherin antibody and 0.4 μ M DETANONOate (F). DETA-NONOate (C) and N-cadherin (D) increase cell adhesion and decrease neurosphere formation. Panel G shows quantitative data of neurosphere formation culture in the different treatment groups for 7 days in growth medium. Scale bar=200 μ m, F.

    Journal: Neuroscience

    Article Title: N-CADHERIN MEDIATES NITRIC OXIDE-INDUCED NEUROGENESIS IN YOUNG AND RETIRED BREEDER NEUROSPHERES

    doi: 10.1016/j.neuroscience.2006.02.064

    Figure Lengend Snippet: N-cadherin regulates neurosphere formation and cell adhesion. Panel A shows quantitative data of N-cadherin mRNA expression in young and retired breeder rat SVZ neurospheres treated with or without DETA-NONOate for 7 days. Panels B–F show young adult neurosphere formation in control (B), treated with 0.4 μ M DETANONOate (C), treated with N-cadherin (5 μ g/cm 2 ) alone (D), treated with anti-N-cadherin antibody (80 ng/ml) alone (E), treated with anti-N-cadherin antibody and 0.4 μ M DETANONOate (F). DETA-NONOate (C) and N-cadherin (D) increase cell adhesion and decrease neurosphere formation. Panel G shows quantitative data of neurosphere formation culture in the different treatment groups for 7 days in growth medium. Scale bar=200 μ m, F.

    Article Snippet: To further investigate whether N-cadherin participates in DETA-NONOate-induced neuronal differentiation, N-cadherin (5 μ g/cm2 ) (recombinant human N-cadherin/Fc chimera, R & D System) and neutralized anti-N-cadherin antibody (monoclonal anti-N-cadherin/A-CAM; Sigma, St. Louis, MO, USA) treatment was employed.

    Techniques: Expressing

    N-cadherin regulates SVZ neurosphere β -catenin mRNA expression, cell differentiation and neurite outgrowth. Panels A–D show quantitative data of TUJ1 (A for retired breeder neurosphere; C for young adult neurosphere), β -catenin (B for retired breeder neurosphere; D for young adult neurosphere) mRNA expression in young and retired breeder neurosphere treated with or without DETA-NONOate, N-cadherin, anti-N-cadherin or anti-N-cadherin with DETA-NONOate. Panel E shows quantitative data of GFAP mRNA expression in young neurospheres treated with or without DETA-NONOate and anti-N-cadherin. Panel F shows quantitative data of TUJ1 immunostaining positive cell neurite outgrowth in young adult neurosphere culture.

    Journal: Neuroscience

    Article Title: N-CADHERIN MEDIATES NITRIC OXIDE-INDUCED NEUROGENESIS IN YOUNG AND RETIRED BREEDER NEUROSPHERES

    doi: 10.1016/j.neuroscience.2006.02.064

    Figure Lengend Snippet: N-cadherin regulates SVZ neurosphere β -catenin mRNA expression, cell differentiation and neurite outgrowth. Panels A–D show quantitative data of TUJ1 (A for retired breeder neurosphere; C for young adult neurosphere), β -catenin (B for retired breeder neurosphere; D for young adult neurosphere) mRNA expression in young and retired breeder neurosphere treated with or without DETA-NONOate, N-cadherin, anti-N-cadherin or anti-N-cadherin with DETA-NONOate. Panel E shows quantitative data of GFAP mRNA expression in young neurospheres treated with or without DETA-NONOate and anti-N-cadherin. Panel F shows quantitative data of TUJ1 immunostaining positive cell neurite outgrowth in young adult neurosphere culture.

    Article Snippet: To further investigate whether N-cadherin participates in DETA-NONOate-induced neuronal differentiation, N-cadherin (5 μ g/cm2 ) (recombinant human N-cadherin/Fc chimera, R & D System) and neutralized anti-N-cadherin antibody (monoclonal anti-N-cadherin/A-CAM; Sigma, St. Louis, MO, USA) treatment was employed.

    Techniques: Expressing, Cell Differentiation, Immunostaining

    CD148 strengthens cell-cell adhesion in A431D/E-cadherin WT, but not A431D or A431D/E-cadherin 764AAA, cells. Effects of CD148 in cell-cell adhesion were assessed by a hanging drop assay. Images show representative data of ten independent experiments. CD148 WT, but not CS, remarkably increases the cell-cell adhesion in A431D/E-cadherin WT cells, while it shows no effects in A431D or A431D/E-cadherin 764 AAA cells.

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 strengthens cell-cell adhesion in A431D/E-cadherin WT, but not A431D or A431D/E-cadherin 764AAA, cells. Effects of CD148 in cell-cell adhesion were assessed by a hanging drop assay. Images show representative data of ten independent experiments. CD148 WT, but not CS, remarkably increases the cell-cell adhesion in A431D/E-cadherin WT cells, while it shows no effects in A431D or A431D/E-cadherin 764 AAA cells.

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques:

    CD148 promotes E-cadherin contact formation with an increase in Rac1 activity. A) Effects of CD148 in E-cadherin contact formation were examined by a calcium-switch assay and immunofluorescence staining. CD148 WT, but not CS, promotes E-cadherin contact formation in A431D/E-cadherin WT cells, as evidenced by the expanded and intense E-cadherin distribution (left panels), while CD148 WT shows no effects in A431D/E-cadherin 764 AAA cells (right panels). B) Activities of Rac1, Cdc42, and RhoA were measured in the condition of calcium switch assay. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by the pull-down assays and/or immunoblotting described in the “ Materials and Methods ” (left panels). The activity was normalized to total amount of protein using densitometry and expressed as fold change (right panels). The data show means ± SEM of quadruplicate determinations. **P

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 promotes E-cadherin contact formation with an increase in Rac1 activity. A) Effects of CD148 in E-cadherin contact formation were examined by a calcium-switch assay and immunofluorescence staining. CD148 WT, but not CS, promotes E-cadherin contact formation in A431D/E-cadherin WT cells, as evidenced by the expanded and intense E-cadherin distribution (left panels), while CD148 WT shows no effects in A431D/E-cadherin 764 AAA cells (right panels). B) Activities of Rac1, Cdc42, and RhoA were measured in the condition of calcium switch assay. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by the pull-down assays and/or immunoblotting described in the “ Materials and Methods ” (left panels). The activity was normalized to total amount of protein using densitometry and expressed as fold change (right panels). The data show means ± SEM of quadruplicate determinations. **P

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Activity Assay, Immunofluorescence, Staining

    CD148 associates less with p120-uncoupled E-cadherin. CD148 was immunoprecipitated from A431D/E-cadherin WT or A431D/E-cadherin 764 AAA cells. Species-matched IgG was used as a control. The immunocomplexes were immunoblotted for E-cadherin and the amounts of CD148 were assessed by reprobing the membranes with anti-CD148. A ratio of E-cadherin to CD148 was quantified by densitometry. Data are representative of four independent experiments. Both CD148 WT and CS associate with wild-type E-cadherin, while CD148 association with p120-uncoupled E-cadherin is relatively limited.

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 associates less with p120-uncoupled E-cadherin. CD148 was immunoprecipitated from A431D/E-cadherin WT or A431D/E-cadherin 764 AAA cells. Species-matched IgG was used as a control. The immunocomplexes were immunoblotted for E-cadherin and the amounts of CD148 were assessed by reprobing the membranes with anti-CD148. A ratio of E-cadherin to CD148 was quantified by densitometry. Data are representative of four independent experiments. Both CD148 WT and CS associate with wild-type E-cadherin, while CD148 association with p120-uncoupled E-cadherin is relatively limited.

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Immunoprecipitation

    Introduction of CD148 forms to A431D and A431D/E-cadherin cells and its effects for E-cadherin and catenin expression and complex formation. A) Wild-type (WT) or catalytically inactive (CS) CD148 was stably introduced to A431D cells lacking classical cadherins [E-cad (−)] or expressing wild-type (WT) or p120-uncoupled mutant (764AAA) E-cadherin. The expression levels of CD148 in these cells were examined by immunoblotting (upper panel) and flow cytometry (lower panel). The loading was assessed by reblotting the membrane for β-actin. B) The levels of E-cadherin, p120, and β-catenin in nearly confluent CD148 stable cells were assessed by immunoblotting, comparing with CD148-negative cells (upper panels). The formation of E-cadherin/catenin complex was assessed by co-immunoprecipitation with E-cadherin (lower panels). Note: The association of E-cadherin with p120 is not observed in A431D/E-cadherin 764 AAA cells.

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: Introduction of CD148 forms to A431D and A431D/E-cadherin cells and its effects for E-cadherin and catenin expression and complex formation. A) Wild-type (WT) or catalytically inactive (CS) CD148 was stably introduced to A431D cells lacking classical cadherins [E-cad (−)] or expressing wild-type (WT) or p120-uncoupled mutant (764AAA) E-cadherin. The expression levels of CD148 in these cells were examined by immunoblotting (upper panel) and flow cytometry (lower panel). The loading was assessed by reblotting the membrane for β-actin. B) The levels of E-cadherin, p120, and β-catenin in nearly confluent CD148 stable cells were assessed by immunoblotting, comparing with CD148-negative cells (upper panels). The formation of E-cadherin/catenin complex was assessed by co-immunoprecipitation with E-cadherin (lower panels). Note: The association of E-cadherin with p120 is not observed in A431D/E-cadherin 764 AAA cells.

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Expressing, Stable Transfection, Mutagenesis, Flow Cytometry, Cytometry, Immunoprecipitation

    Effects of CD148 in E-cadherin distribution. Immunofluorescence localization of CD148 (green), E-cadherin (red), and p120 (purple) were examined in CD148 WT or CS-introduced A431D/E-cadherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells and compared with CD148-negative cells. Lower panels show a higher magnification of E-cadherin immunofluorescence. Wild-type E-cadherin is more broadly distributed at cell junctions in CD148 WT-introduced cells (arrowheads in left panels), while the distribution of p120-uncoupled E-cadherin is unaltered by CD148 WT introduction (right panels).

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: Effects of CD148 in E-cadherin distribution. Immunofluorescence localization of CD148 (green), E-cadherin (red), and p120 (purple) were examined in CD148 WT or CS-introduced A431D/E-cadherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells and compared with CD148-negative cells. Lower panels show a higher magnification of E-cadherin immunofluorescence. Wild-type E-cadherin is more broadly distributed at cell junctions in CD148 WT-introduced cells (arrowheads in left panels), while the distribution of p120-uncoupled E-cadherin is unaltered by CD148 WT introduction (right panels).

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Immunofluorescence

    CD148 increases Rac1 activity in the condition of a hanging drop assay. A and B) CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 increases Rac1 activity in the condition of a hanging drop assay. A and B) CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Activity Assay

    CD148 dephosphorylates p120 and β-catenin in vitro . A) A431D/E-cadherin WT and A431D cells were treated with (+) or without (−) pervandadate (PV) and cell lysates were incubated with GST or GST-CD148 (WT, DA) proteins. GST-protein complex were pulled-down using glutathione beads and the protein interactions were examined by immunoblotting (left panels). Vandadate (VO4) competition was also assessed (right panels). Substrate-trapping (DA), but not WT, form of CD148 binds to p120 and β-catenin in a phosphorylation dependent manner and these interactions are blocked by vanadate (VO4). B) CD148 dephosphorylation of E-cadherin, p120, and β-catenin was assessed in vitro . E-cadherin, p120, and β-catenin were immunoprecipitated from the pervanadate (PV)-treated or untreated A431D/E-cadherin WT cells. The immunoprecipitates were incubated with GST or GST-CD148 proteins and its effects were assessed by immunoblotting with a pY20 phosphotyrosine antibody (pY) (left panels). The amount of protein was assessed by reprobing the membranes with specific antibodies. Vanadate (VO4) competition was also assessed (right panels). CD148 WT, but not CS, dephosphorylates p120 and β-catenin in a dose dependent manner, while its effects for E-cadherin are limited. CD148 dephosphorylation of p120 and β-catenin is blocked by vanadate (VO 4 ).

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 dephosphorylates p120 and β-catenin in vitro . A) A431D/E-cadherin WT and A431D cells were treated with (+) or without (−) pervandadate (PV) and cell lysates were incubated with GST or GST-CD148 (WT, DA) proteins. GST-protein complex were pulled-down using glutathione beads and the protein interactions were examined by immunoblotting (left panels). Vandadate (VO4) competition was also assessed (right panels). Substrate-trapping (DA), but not WT, form of CD148 binds to p120 and β-catenin in a phosphorylation dependent manner and these interactions are blocked by vanadate (VO4). B) CD148 dephosphorylation of E-cadherin, p120, and β-catenin was assessed in vitro . E-cadherin, p120, and β-catenin were immunoprecipitated from the pervanadate (PV)-treated or untreated A431D/E-cadherin WT cells. The immunoprecipitates were incubated with GST or GST-CD148 proteins and its effects were assessed by immunoblotting with a pY20 phosphotyrosine antibody (pY) (left panels). The amount of protein was assessed by reprobing the membranes with specific antibodies. Vanadate (VO4) competition was also assessed (right panels). CD148 WT, but not CS, dephosphorylates p120 and β-catenin in a dose dependent manner, while its effects for E-cadherin are limited. CD148 dephosphorylation of p120 and β-catenin is blocked by vanadate (VO 4 ).

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: In Vitro, Incubation, De-Phosphorylation Assay, Immunoprecipitation

    A Hypothetical model depicting CD148 regulation of E-cadherin cell-cell adhesion. CD148 dephosphorylates p120 and β-catenin in E-cadherin contacts. It also dephosphorylates the suppressive tyrosine residue (Y529) in Src, increasing Src activity and possibly enhancing the phosphorylation of Y228 in p120. These signaling events increase Rac1 activity and promote the expansion of contact zones and the stabilization of cadherin complexes, resulting in stronger E-cadherin cell-cell adhesion.

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: A Hypothetical model depicting CD148 regulation of E-cadherin cell-cell adhesion. CD148 dephosphorylates p120 and β-catenin in E-cadherin contacts. It also dephosphorylates the suppressive tyrosine residue (Y529) in Src, increasing Src activity and possibly enhancing the phosphorylation of Y228 in p120. These signaling events increase Rac1 activity and promote the expansion of contact zones and the stabilization of cadherin complexes, resulting in stronger E-cadherin cell-cell adhesion.

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Activity Assay

    CD148 knockdown reduces cell-cell adhesion and E-cadherin contacts accompanied by a decrease in Rac1 activity in A431 cells. A) A431 cells was infected with a lentivirus encoding CD148-targeting or scrambled shRNA. Cells were harvested at 72 h after infection and the expression of CD148, E-cadherin, p120, and β- catenin was assessed by immunoblot analysis. Equal loading was confirmed by reblotting the membrane for β-actin. The targeting shRNA reduces CD148 expression (∼65%) without altering the E-cadherin, p120, and β-catenin expression. B) CD148 knock-down cells and the control cells treated with scrambled shRNA were subjected to a handing drop assay. Data are representative of five independent experiments. CD148 knockdown reduces cell aggregation in A431 cells. C and D) CD148 knock-down and control cells were subjected to a calcium switch assay and were immunostained for E-cadherin (panel C). Rac1 activity in these cells was also assessed (panel D). Representative results of four independent experiments are shown. The data show means ± SEM of quadruplicate determinations. **P

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 knockdown reduces cell-cell adhesion and E-cadherin contacts accompanied by a decrease in Rac1 activity in A431 cells. A) A431 cells was infected with a lentivirus encoding CD148-targeting or scrambled shRNA. Cells were harvested at 72 h after infection and the expression of CD148, E-cadherin, p120, and β- catenin was assessed by immunoblot analysis. Equal loading was confirmed by reblotting the membrane for β-actin. The targeting shRNA reduces CD148 expression (∼65%) without altering the E-cadherin, p120, and β-catenin expression. B) CD148 knock-down cells and the control cells treated with scrambled shRNA were subjected to a handing drop assay. Data are representative of five independent experiments. CD148 knockdown reduces cell aggregation in A431 cells. C and D) CD148 knock-down and control cells were subjected to a calcium switch assay and were immunostained for E-cadherin (panel C). Rac1 activity in these cells was also assessed (panel D). Representative results of four independent experiments are shown. The data show means ± SEM of quadruplicate determinations. **P

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Activity Assay, Infection, shRNA, Expressing

    CD148 regulates the tyrosine phosphorylation of p120, β-catenin, and Src upon E-cadherin engagement. Effects of CD148 in the cadherin adhesion-associated tyrosine phosphorylation of p120, β-catenin, and Src were assessed by a calcium-switch assay and immunoblot analysis using A431D/E-caherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells. For p120 and β-catenin, tyrosine phosphorylation of p120 and β-catenin that were co-immunoprecipitated with E-cadherin was assessed by immunoblotting. In A431D/E-cadherin 764 AAA cells, p120 was immunoprecipitated. The membranes were reprobed with p120, β-catenin and Src antibodies and a ratio of phosphorylated to total protein was quantified by densitometry. Data are representative of five independent experiments. CD148 WT, but not CS, reduces the tyrosine phosphorylation of p120, β-catenin, and Src (Y529) upon E-cadherin engagement in A431D/E-cadherin WT cells, while it increases the phosphorylation of Y228 (a Src site) in p120. These effects are not observed in A431D/E-cadherin 764 AAA cells.

    Journal: PLoS ONE

    Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

    doi: 10.1371/journal.pone.0112753

    Figure Lengend Snippet: CD148 regulates the tyrosine phosphorylation of p120, β-catenin, and Src upon E-cadherin engagement. Effects of CD148 in the cadherin adhesion-associated tyrosine phosphorylation of p120, β-catenin, and Src were assessed by a calcium-switch assay and immunoblot analysis using A431D/E-caherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells. For p120 and β-catenin, tyrosine phosphorylation of p120 and β-catenin that were co-immunoprecipitated with E-cadherin was assessed by immunoblotting. In A431D/E-cadherin 764 AAA cells, p120 was immunoprecipitated. The membranes were reprobed with p120, β-catenin and Src antibodies and a ratio of phosphorylated to total protein was quantified by densitometry. Data are representative of five independent experiments. CD148 WT, but not CS, reduces the tyrosine phosphorylation of p120, β-catenin, and Src (Y529) upon E-cadherin engagement in A431D/E-cadherin WT cells, while it increases the phosphorylation of Y228 (a Src site) in p120. These effects are not observed in A431D/E-cadherin 764 AAA cells.

    Article Snippet: The cells were immunostained with VE-cadherin (Cadherin 5, BD biosciences, San Jose, CA) or HA (mouse monoclonal, Covance, Princeton, NJ) antibodie s followed by incubation with a secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen Corporation, Carlsbad, CA).

    Techniques: Immunoprecipitation

    Effect of E-cadherin overexpression on cell dimensions. Hearts from adult rabbits were excised and single cells enzymatically isolated using a Langendorff perfusion system. Isolated single adult rabbit cells and H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ Cell dimensions were measured using a graticule in the eye-piece of a microscope. (A) Transduction of H9c2 cells with AD:E-cadherin resulted in significantly decreased cell diameter ( n = 50/group). (B) Infection of rabbit myocytes with Ad:E-cadherin also resulted in cells with a significantly decreased cell length and width resulting in an overall decrease in cell volume ( n = 50/group).

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

    doi: 10.1016/j.yjmcc.2010.01.017

    Figure Lengend Snippet: Effect of E-cadherin overexpression on cell dimensions. Hearts from adult rabbits were excised and single cells enzymatically isolated using a Langendorff perfusion system. Isolated single adult rabbit cells and H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ Cell dimensions were measured using a graticule in the eye-piece of a microscope. (A) Transduction of H9c2 cells with AD:E-cadherin resulted in significantly decreased cell diameter ( n = 50/group). (B) Infection of rabbit myocytes with Ad:E-cadherin also resulted in cells with a significantly decreased cell length and width resulting in an overall decrease in cell volume ( n = 50/group).

    Article Snippet: Tissue sections were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control (Dako, Denmark) followed by detection with biotinylated universal secondary antibody (1/200), ABC Kit and standard diaminobenzidine staining.

    Techniques: Over Expression, Isolation, Infection, Microscopy, Transduction

    Localisation of β-catenin following E-cadherin overexpression. H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ and levels and localisation of N-cadherin and β-catenin were analysed. (A) Immunocytochemistry of H9c2 cells overexpressing E-cadherin showing up-regulation of β-catenin both in the cytoplasm but also markedly at the cell-to-cell junctions (indicated by the arrows). Many of the infected cells also contained two or more nuclei (B) H9c2 cells infected with either Ad:E-cadherin or Ad: LacZ . Cells were lysed in RIPA buffer and undergone immunoprecipitation with either β-catenin or IgG control antibody. Western blot showing that much of the E-cadherin co-precipitates and therefore co-localises with β-catenin. (C) Immunohistochemical localisation of β-catenin in the hearts of 12-week old SHRSP and WKY controls. There appears to be selective localisation of β-catenin at the intercalated disc of the cell membrane in the SHRSP hearts (indicated by the arrows, scale = 10 µm). (D) Immunohistochemical staining of WKY and SHRSP hearts showing localisation of β-catenin to the intercalated disc similar to the adherens junction marker connexin 43 (scale = 10 µm).

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

    doi: 10.1016/j.yjmcc.2010.01.017

    Figure Lengend Snippet: Localisation of β-catenin following E-cadherin overexpression. H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ and levels and localisation of N-cadherin and β-catenin were analysed. (A) Immunocytochemistry of H9c2 cells overexpressing E-cadherin showing up-regulation of β-catenin both in the cytoplasm but also markedly at the cell-to-cell junctions (indicated by the arrows). Many of the infected cells also contained two or more nuclei (B) H9c2 cells infected with either Ad:E-cadherin or Ad: LacZ . Cells were lysed in RIPA buffer and undergone immunoprecipitation with either β-catenin or IgG control antibody. Western blot showing that much of the E-cadherin co-precipitates and therefore co-localises with β-catenin. (C) Immunohistochemical localisation of β-catenin in the hearts of 12-week old SHRSP and WKY controls. There appears to be selective localisation of β-catenin at the intercalated disc of the cell membrane in the SHRSP hearts (indicated by the arrows, scale = 10 µm). (D) Immunohistochemical staining of WKY and SHRSP hearts showing localisation of β-catenin to the intercalated disc similar to the adherens junction marker connexin 43 (scale = 10 µm).

    Article Snippet: Tissue sections were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control (Dako, Denmark) followed by detection with biotinylated universal secondary antibody (1/200), ABC Kit and standard diaminobenzidine staining.

    Techniques: Over Expression, Infection, Immunocytochemistry, Immunoprecipitation, Western Blot, Immunohistochemistry, Staining, Marker

    Cadherin and catenin expression in the SHRSP and WKY rat hearts. Western blot analysis of heart extracts from 12-week old SHRSP and WKY animals (shown as 3 of each, n = 9). (A) Western blot analysis from heart protein homogenates prepared from animals 12 weeks of age indicated that the levels of β-, γ- and α-catenin were all unchanged between the control and the SHRSP animals. (B) Levels of T-, N- and R-cadherin also remained unchanged, however we observed a marked up-regulated level of E-cadherin from low levels in the WKY to readily detectable levels in the SHRSP. (C) Further analysis of heart homogenates from 6, 12 and 18 week old animals showed these cadherin levels were up-regulated from as early as 6 weeks of age through to 18 weeks.

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

    doi: 10.1016/j.yjmcc.2010.01.017

    Figure Lengend Snippet: Cadherin and catenin expression in the SHRSP and WKY rat hearts. Western blot analysis of heart extracts from 12-week old SHRSP and WKY animals (shown as 3 of each, n = 9). (A) Western blot analysis from heart protein homogenates prepared from animals 12 weeks of age indicated that the levels of β-, γ- and α-catenin were all unchanged between the control and the SHRSP animals. (B) Levels of T-, N- and R-cadherin also remained unchanged, however we observed a marked up-regulated level of E-cadherin from low levels in the WKY to readily detectable levels in the SHRSP. (C) Further analysis of heart homogenates from 6, 12 and 18 week old animals showed these cadherin levels were up-regulated from as early as 6 weeks of age through to 18 weeks.

    Article Snippet: Tissue sections were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control (Dako, Denmark) followed by detection with biotinylated universal secondary antibody (1/200), ABC Kit and standard diaminobenzidine staining.

    Techniques: Expressing, Western Blot

    Altered E-cadherin expression. (A) Taqman gene expression analysis of RNA extracted from the hearts of 12-week old SHRSP and WKY animals. Data shows a significant increase in expression of E-cadherin in the SHRSP group and is shown as relative fold change ( n = 6). (B) Genome organisation of E-cadherin. Sequence analysis identified a nucleotide change for SNP1 located in the non-coding region of exon 1.

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

    doi: 10.1016/j.yjmcc.2010.01.017

    Figure Lengend Snippet: Altered E-cadherin expression. (A) Taqman gene expression analysis of RNA extracted from the hearts of 12-week old SHRSP and WKY animals. Data shows a significant increase in expression of E-cadherin in the SHRSP group and is shown as relative fold change ( n = 6). (B) Genome organisation of E-cadherin. Sequence analysis identified a nucleotide change for SNP1 located in the non-coding region of exon 1.

    Article Snippet: Tissue sections were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control (Dako, Denmark) followed by detection with biotinylated universal secondary antibody (1/200), ABC Kit and standard diaminobenzidine staining.

    Techniques: Expressing, Sequencing

    Adenovirus mediated in vitro overexpression of E-cadherin. Isolated single adult rabbit cells or H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ . (A) Western blot from extracts of H9c2 cells infected with E-cadherin showing increasing levels of E-cadherin from differing multiplicities of infection and the GAPDH loading control (B) Immunocytochemical levels of E-cadherin in H9c2 cells infected with either Ad:E-cadherin or Ad: LacZ . Successful infection of E-cadherin into the H9c2 cell-line can be observed at low and high levels of infection (scale = 25 µm).

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

    doi: 10.1016/j.yjmcc.2010.01.017

    Figure Lengend Snippet: Adenovirus mediated in vitro overexpression of E-cadherin. Isolated single adult rabbit cells or H9c2 cells were infected with either Ad:E-cadherin or Ad: LacZ . (A) Western blot from extracts of H9c2 cells infected with E-cadherin showing increasing levels of E-cadherin from differing multiplicities of infection and the GAPDH loading control (B) Immunocytochemical levels of E-cadherin in H9c2 cells infected with either Ad:E-cadherin or Ad: LacZ . Successful infection of E-cadherin into the H9c2 cell-line can be observed at low and high levels of infection (scale = 25 µm).

    Article Snippet: Tissue sections were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control (Dako, Denmark) followed by detection with biotinylated universal secondary antibody (1/200), ABC Kit and standard diaminobenzidine staining.

    Techniques: In Vitro, Over Expression, Isolation, Infection, Western Blot

    Histological analysis of hearts from the SHRSP and WKY. Histological analysis was carried out on the hearts of 12-week old WKY and SHRSP animals. (A) There appeared to be no difference in levels of N-cadherin between the SHRSP animals and the WKY controls. Immunohistochemistry of tissue sections for E-cadherin demonstrated up-regulated levels of E-cadherin in the SHRSP compared to the WKY control (scale = 25 µm). (B) Immunohistochemical staining of SHRSP hearts showing localisation of E-cadherin to the intercalated disc similar to the adherens junction marker connexin 43 (scale = 25 µm).

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Dysregulation of cadherins in the intercalated disc of the spontaneously hypertensive stroke-prone rat

    doi: 10.1016/j.yjmcc.2010.01.017

    Figure Lengend Snippet: Histological analysis of hearts from the SHRSP and WKY. Histological analysis was carried out on the hearts of 12-week old WKY and SHRSP animals. (A) There appeared to be no difference in levels of N-cadherin between the SHRSP animals and the WKY controls. Immunohistochemistry of tissue sections for E-cadherin demonstrated up-regulated levels of E-cadherin in the SHRSP compared to the WKY control (scale = 25 µm). (B) Immunohistochemical staining of SHRSP hearts showing localisation of E-cadherin to the intercalated disc similar to the adherens junction marker connexin 43 (scale = 25 µm).

    Article Snippet: Tissue sections were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control (Dako, Denmark) followed by detection with biotinylated universal secondary antibody (1/200), ABC Kit and standard diaminobenzidine staining.

    Techniques: Immunohistochemistry, Staining, Marker