anti-e-cadherin Search Results


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  • 98
    Thermo Fisher anti e cadherin
    LPA induces MMP-9-dependent <t>E-cadherin</t> ectodomain shedding. (a) OVCA429 and OVCA433 cells were treated with LPA (0, 20, or 80 μ M, as indicated) and conditioned media evaluated for MMP expression by gelatin zymography. (b) Cells were treated with LPA (20 μ M) in the presence or absence of anti-MMP-9 function blocking antibody (10 μ g/mL). Conditioned media were subjected to immunoprecipitation with <t>anti-E-cadherin</t> antibodies, and precipitates were western blotted with a second E-cadherin antibody (Zymed, 1 : 1,000) followed by peroxidase-conjugated secondary antibody (1 : 5,000) and enhanced chemiluminescence detection. The lower panel shows densitometric quantitation of replicate western blots.
    Anti E Cadherin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti e cadherin
    Cl-Amidine treatment increases <t>E-cadherin</t> expression in MCF10DCIS.com xenograft tumors. a PAS staining of representative mammary tissue sections of MCF10DCIS.com xenograft mice following either vehicle alone treatment (PBS) or treatment with Cl-Amidine. PAS stained sections were prepared and imaged using the bright field optics of the Axiophot inverted microscope. Scale bar = 200 μm. b <t>Anti-E-cadherin</t> IF staining of tissue sections from MCF10DCIS.com xenograft tumors following vehicle alone treatment (PBS) or with Cl-Amidine treatment. Scale bar = 50 μm
    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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    Abcam rat anti e cadherin
    Cl-Amidine treatment increases <t>E-cadherin</t> expression in MCF10DCIS.com xenograft tumors. a PAS staining of representative mammary tissue sections of MCF10DCIS.com xenograft mice following either vehicle alone treatment (PBS) or treatment with Cl-Amidine. PAS stained sections were prepared and imaged using the bright field optics of the Axiophot inverted microscope. Scale bar = 200 μm. b <t>Anti-E-cadherin</t> IF staining of tissue sections from MCF10DCIS.com xenograft tumors following vehicle alone treatment (PBS) or with Cl-Amidine treatment. Scale bar = 50 μm
    Rat Anti E Cadherin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti e cadherin
    PKD1 conserves the epithelial phenotype in normal mammary gland cells. A: NMuMG cells were either left untreated or were treated with TGFβ1 (10 ng/ml) for 48 hours. Cell morphology was photographed (bar is 200 µm) and cells were harvested and analyzed for expression of epithelial <t>(E-cadherin,</t> cytokeratin) and mesenchymal (N-cadherin) markers by Western blotting with anti-N-cadherin, <t>anti-E-cadherin,</t> or anti-cytokeratin antibodies. Staining for actin (anti-actin) served as a loading control. B: NMuMG cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Endogenous PKD1 was immunoprecipitated (anti-PKD1) and analyzed for phosphorylation at its activation loop that correlates with its activity (anti-pS738/742-PKD), or samples were control stained for total PKD1 (anti-PKD1). C: Cells were stimulated with PMA (100 nM, 10 min), EGF (50 ng/ml, 10 min), Bradykinin (0.5 µg/ml, 10 min) or left untreated. Endogenous PKD1 was immunoprecipitated and subjected to an in vitro kinase assay using PKD substrate peptide. PKD1 activity is depicted relative to PMA-activated PKD1 (maximum activation). Equal immunoprecipitation was controlled by SDS-PAGE and immunoblot (anti-PKD1). D: NMuMG cells were either transfected with control vector or with active PKD1 (PKD1.CA, PKD1.S738E.S742E). 24 hours after transfection, cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Lysates were analyzed for expression of N-cadherin, E-cadherin, expression of PKD1, or actin as a loading control. E: NMuMG cells were stably-transfected with vector control, wildtype PKD1 or kinase-dead PKD1.K612W (PKD1.KD) Cell morphology was analyzed by brightfield microscopy (bar is 200 µm). Expression of endogenous and overexpressed PKD1 was determined by Western blot analysis using an anti-PKD1 antibody. Immunoblotting for actin (anti-actin) served as loading control.
    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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    93
    Agilent technologies anti e cadherin
    IL-11 Expression is Correlated with Progression of Human Cancers. a Specificity of anti-IL-11 antibody used in the study. MDA-MB-231 cells were treated with control or three different siRNAs against human Il11 , and IL-11 expression in cell lysates was analyzed by Western blotting with anti-IL-11 and anti-tubulin antibodies. Results are representative of two independent experiments. b-d Adenomas (n = 10), early (n = 10) and advanced (n = 10) colorectal cancers were stained with anti-IL-11 antibody ( b ). Representative staining of respective tumors and adjacent normal tissues. Scale bars, 100 μm. Areas ( c ) and intensities ( d ) of IL-11 + signals were calculated as in the methods. Results are mean ± SE. e Tissues of advanced colorectal cancers were stained with anti-vimentin, anti-CD45, or <t>anti-E-cadherin</t> antibodies along with anti-IL-11 antibody (n = 3). Right panels are enlarged images of the white box in the left panels. Signaling intensities stained with the indicated antibodies on the white arrows were calculated and plotted in the right panels. Scale bars, 100 μm. Statistical significance was determined by Mann-Whitney U test ( c, d ). **p
    Anti E Cadherin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti e cadherin
    Urothelium-specific and inducible expression of FGFR3b-S243C mutant in transgenic mice. ( A ) Transgene constructs. Line 1 (L1) represented a previously generated transgenic mouse line in which the 3.6-kB mouse uroplakin II (UPII) promoter drove the expression of a modified reverse tetracycline transactivator (rtTA-M2). pA, poly A signal. Line 2 (L2) was a newly generated transgenic mouse line in which the tetracycline responsive elements (TRE) drove the expression of hemagglutinin (HA)-tagged mouse FGFR3b-S243C mutant cDNA. S1, S2 and AS were oligonucleotide primers used for RT-PCR and sequencing (see panels ( B,C )). ( B ) RT-PCR and Western blotting of total RNAs and total proteins, respectively, from the double transgenic mice harboring both UPII-rtTA-M2 and TRE-FGFR3b-S243C transgenes. UI, un-induced mice (i.e., mice placed on a regular diet for 14 days); I, induced mice (i.e., mice placed on doxycycline-containing diet for 14 days). Three representative mice were shown for each condition. Note the specific detection of the FGFR3b-S243C on RNA and protein levels only in the induced mice. ( C ) Direct sequencing of the RT-PCR products from the S2/AS primer amplification showed the un-mutated codon 243 (TCC encoding serine (S)) in un-induced double transgenic mice, representing the endogenous wild-type FGFR3b; and the mutated codon 243 (TGC encoding cysteine ( C )) in induced mice, representing the transgene product. Only three codons were shown and the rest of the regions were all identical. ( D ) Double immunofluorescent staining using anti-HA and <t>anti-E-cadherin</t> (Ecad) showing the lack of expression of the FGFRb-S243C in un-induced mice or the strong expression of the mutant in induced mice (induction for 14 days). Also note the presence of both cytoplasmic and surface membrane staining of FGFR3b-S243C (overlapped with anti-Ecad; highlighted in dotted boxes). Magnification in ( D ), 200x.
    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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    92
    TaKaRa anti e cadherin
    TGF-β/TGFβR signaling is involved in podoplanin-induced epithelial-mesenchymal transition in UM-UC-5 cells. ( a – c ) UM-UC-5 cells were treated with or without TGF-β1 neutralizing mAb (1D11 mAb) or TGFβR inhibitors (LY2157299 or SB431542) for 2 h, followed by incubation with supernatants of UM-UC-5-platelet reactants for 48 h. Morphological and physiological changes in treated cells were examined by immunoblotting ( a ), immunofluorescence staining ( b ) and invasion assay using a matrigel-coated transwell chambers ( c ). ( a ) Cell lysates were immunoblotted with antibodies to <t>N-cadherin,</t> claudin-1, podoplanin (PDPN, clone D2-40) and TopoIIβ. ( b ) Cells were stained for <t>anti-E-cadherin</t> (green), F-actin (red; phalloidin) and nuclear DNA (blue; Hoechst 33342). Scale bars represent 50 μm. ( c ) Cells were either left untreated or treated with supernatants of UM-UC-5-platelet reactants for 48 h. Next, 5 × 10 4 UM-UC-5 cells were added to the upper chambers of matrigel-overlaid membranes. After incubation for an additional 48 h at 37 °C, cells migrating through the membranes were fixed and stained with crystal violet (lower panels; scale bars represent 200 μm). Optical density (OD) of crystal violet extracted from cells was measured at 540 nm and presented as a percentage of the OD values of control cells. All data are shown as means ± standard deviation (SD, n = 8). **P
    Anti E Cadherin, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend anti e cadherin
    TGF-β/TGFβR signaling is involved in podoplanin-induced epithelial-mesenchymal transition in UM-UC-5 cells. ( a – c ) UM-UC-5 cells were treated with or without TGF-β1 neutralizing mAb (1D11 mAb) or TGFβR inhibitors (LY2157299 or SB431542) for 2 h, followed by incubation with supernatants of UM-UC-5-platelet reactants for 48 h. Morphological and physiological changes in treated cells were examined by immunoblotting ( a ), immunofluorescence staining ( b ) and invasion assay using a matrigel-coated transwell chambers ( c ). ( a ) Cell lysates were immunoblotted with antibodies to <t>N-cadherin,</t> claudin-1, podoplanin (PDPN, clone D2-40) and TopoIIβ. ( b ) Cells were stained for <t>anti-E-cadherin</t> (green), F-actin (red; phalloidin) and nuclear DNA (blue; Hoechst 33342). Scale bars represent 50 μm. ( c ) Cells were either left untreated or treated with supernatants of UM-UC-5-platelet reactants for 48 h. Next, 5 × 10 4 UM-UC-5 cells were added to the upper chambers of matrigel-overlaid membranes. After incubation for an additional 48 h at 37 °C, cells migrating through the membranes were fixed and stained with crystal violet (lower panels; scale bars represent 200 μm). Optical density (OD) of crystal violet extracted from cells was measured at 540 nm and presented as a percentage of the OD values of control cells. All data are shown as means ± standard deviation (SD, n = 8). **P
    Anti E Cadherin, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank anti e cadherin
    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by <t>E-cadherin</t> blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays <t>anti-E-cadherin</t> immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P
    Anti E Cadherin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex anti e cadherin
    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by <t>E-cadherin</t> blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays <t>anti-E-cadherin</t> immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P
    Anti E Cadherin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Lifespan Biosciences anti e cadherin
    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by <t>E-cadherin</t> blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays <t>anti-E-cadherin</t> immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P
    Anti E Cadherin, supplied by Lifespan Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Epitomics anti e cadherin
    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by <t>E-cadherin</t> blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays <t>anti-E-cadherin</t> immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P
    Anti E Cadherin, supplied by Epitomics, used in various techniques. Bioz Stars score: 93/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Proteintech anti e cadherin
    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by <t>E-cadherin</t> blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays <t>anti-E-cadherin</t> immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P
    Anti E Cadherin, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ventana Medical anti e cadherin
    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by <t>E-cadherin</t> blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays <t>anti-E-cadherin</t> immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P
    Anti E Cadherin, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LPA induces MMP-9-dependent E-cadherin ectodomain shedding. (a) OVCA429 and OVCA433 cells were treated with LPA (0, 20, or 80 μ M, as indicated) and conditioned media evaluated for MMP expression by gelatin zymography. (b) Cells were treated with LPA (20 μ M) in the presence or absence of anti-MMP-9 function blocking antibody (10 μ g/mL). Conditioned media were subjected to immunoprecipitation with anti-E-cadherin antibodies, and precipitates were western blotted with a second E-cadherin antibody (Zymed, 1 : 1,000) followed by peroxidase-conjugated secondary antibody (1 : 5,000) and enhanced chemiluminescence detection. The lower panel shows densitometric quantitation of replicate western blots.

    Journal: Journal of Oncology

    Article Title: Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

    doi: 10.1155/2012/501492

    Figure Lengend Snippet: LPA induces MMP-9-dependent E-cadherin ectodomain shedding. (a) OVCA429 and OVCA433 cells were treated with LPA (0, 20, or 80 μ M, as indicated) and conditioned media evaluated for MMP expression by gelatin zymography. (b) Cells were treated with LPA (20 μ M) in the presence or absence of anti-MMP-9 function blocking antibody (10 μ g/mL). Conditioned media were subjected to immunoprecipitation with anti-E-cadherin antibodies, and precipitates were western blotted with a second E-cadherin antibody (Zymed, 1 : 1,000) followed by peroxidase-conjugated secondary antibody (1 : 5,000) and enhanced chemiluminescence detection. The lower panel shows densitometric quantitation of replicate western blots.

    Article Snippet: Cells were fixed in 4% PFA for 20 minutes at room temperature, followed by immunostaining with anti-E-cadherin (hecd-1 clone, Zymed, San Francisco, CA; 1 : 300) and Alexa-Fluor 488-conjugated secondary antibody (1 : 500).

    Techniques: Expressing, Zymography, Blocking Assay, Immunoprecipitation, Western Blot, Quantitation Assay

    Inhibition of uPA activity does not prevent LPA-induced E-cadherin junction disruption. OVCA429 cells were treated with or without LPA (30 μ M), as indicated, for 18 hours in the presence or absence of the uPA inhibitor designated uPA Stop (2.5 μ M), as indicated and processed for immunofluorescence staining for E-cadherin using anti-E-cadherin ectodomain antibody (1 : 300) and Alexa Fluor 488-conjugated secondary antibody (1 : 500; green). Blue-DAPI-stained nuclei.

    Journal: Journal of Oncology

    Article Title: Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

    doi: 10.1155/2012/501492

    Figure Lengend Snippet: Inhibition of uPA activity does not prevent LPA-induced E-cadherin junction disruption. OVCA429 cells were treated with or without LPA (30 μ M), as indicated, for 18 hours in the presence or absence of the uPA inhibitor designated uPA Stop (2.5 μ M), as indicated and processed for immunofluorescence staining for E-cadherin using anti-E-cadherin ectodomain antibody (1 : 300) and Alexa Fluor 488-conjugated secondary antibody (1 : 500; green). Blue-DAPI-stained nuclei.

    Article Snippet: Cells were fixed in 4% PFA for 20 minutes at room temperature, followed by immunostaining with anti-E-cadherin (hecd-1 clone, Zymed, San Francisco, CA; 1 : 300) and Alexa-Fluor 488-conjugated secondary antibody (1 : 500).

    Techniques: Inhibition, Activity Assay, Immunofluorescence, Staining

    LPA induces MMP-dependent E-cadherin ectodomain shedding. (a, b) OVCA429 or (c, d) OVCA433 cells were treated with LPA (30 μ M) in the presence or absence of the broad-spectrum MMP inhibitor GM6001 (25 μ M) as indicated for 24 hours. Conditioned media were subjected to immunoprecipitation with anti-E-cadherin antibodies, and precipitates were western blotted with a second E-cadherin antibody (Zymed, 1 : 1,000) followed by peroxidase-conjugated secondary antibody (1 : 5,000) and enhanced chemiluminescence detection. Panels (b, d) show densitometric quantitation of replicate western blots.

    Journal: Journal of Oncology

    Article Title: Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

    doi: 10.1155/2012/501492

    Figure Lengend Snippet: LPA induces MMP-dependent E-cadherin ectodomain shedding. (a, b) OVCA429 or (c, d) OVCA433 cells were treated with LPA (30 μ M) in the presence or absence of the broad-spectrum MMP inhibitor GM6001 (25 μ M) as indicated for 24 hours. Conditioned media were subjected to immunoprecipitation with anti-E-cadherin antibodies, and precipitates were western blotted with a second E-cadherin antibody (Zymed, 1 : 1,000) followed by peroxidase-conjugated secondary antibody (1 : 5,000) and enhanced chemiluminescence detection. Panels (b, d) show densitometric quantitation of replicate western blots.

    Article Snippet: Cells were fixed in 4% PFA for 20 minutes at room temperature, followed by immunostaining with anti-E-cadherin (hecd-1 clone, Zymed, San Francisco, CA; 1 : 300) and Alexa-Fluor 488-conjugated secondary antibody (1 : 500).

    Techniques: Immunoprecipitation, Western Blot, Quantitation Assay

    LPA induces E-cadherin junction disruption in EOC cells. (a) Confluent monolayers of OVCA433, OVCA429, or OVCAR3 cells, as indicated, were treated with LPA (20 μ M) for 18–24 hours and processed for immunofluorescence staining for E-cadherin using anti-E-cadherin ectodomain antibody (1 : 300) and Alexa Fluor 488-conjugated secondary antibody (1 : 500; green). Blue-DAPI-stained nuclei. (b) To evaluate the dose-dependence of LPA-induced junction loss, OVCA429 cells were treated with LPA at the concentrations indicated for 24 hours and processed for E-cadherin immunofluorescence (green) as described in (a) above. Blue-DAPI-stained nuclei. (c) Junction loss was quantified by counting the number of cells/field with two remaining E-cadherin immunostained borders in a minimum of 5 fields per treatment (at least 100 cells).

    Journal: Journal of Oncology

    Article Title: Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

    doi: 10.1155/2012/501492

    Figure Lengend Snippet: LPA induces E-cadherin junction disruption in EOC cells. (a) Confluent monolayers of OVCA433, OVCA429, or OVCAR3 cells, as indicated, were treated with LPA (20 μ M) for 18–24 hours and processed for immunofluorescence staining for E-cadherin using anti-E-cadherin ectodomain antibody (1 : 300) and Alexa Fluor 488-conjugated secondary antibody (1 : 500; green). Blue-DAPI-stained nuclei. (b) To evaluate the dose-dependence of LPA-induced junction loss, OVCA429 cells were treated with LPA at the concentrations indicated for 24 hours and processed for E-cadherin immunofluorescence (green) as described in (a) above. Blue-DAPI-stained nuclei. (c) Junction loss was quantified by counting the number of cells/field with two remaining E-cadherin immunostained borders in a minimum of 5 fields per treatment (at least 100 cells).

    Article Snippet: Cells were fixed in 4% PFA for 20 minutes at room temperature, followed by immunostaining with anti-E-cadherin (hecd-1 clone, Zymed, San Francisco, CA; 1 : 300) and Alexa-Fluor 488-conjugated secondary antibody (1 : 500).

    Techniques: Immunofluorescence, Staining

    Inhibition of LPA receptor signaling blocks LPA-induced E-cadherin junction loss. OVCA429 cells were treated with LPA (80 μ M) for 24 hours in the presence or absence of LPARI (20 μ M) or DMSO (vehicle control), as indicated. (a) Conditioned media were examined by gelatin zymography. (b) Cells were processed for E-cadherin immunofluorescence using anti-E-cadherin ectodomain antibody (1 : 300) and Alexa Fluor 488-conjugated secondary antibody (1 : 500; green). Blue-DAPI-stained nuclei.

    Journal: Journal of Oncology

    Article Title: Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

    doi: 10.1155/2012/501492

    Figure Lengend Snippet: Inhibition of LPA receptor signaling blocks LPA-induced E-cadherin junction loss. OVCA429 cells were treated with LPA (80 μ M) for 24 hours in the presence or absence of LPARI (20 μ M) or DMSO (vehicle control), as indicated. (a) Conditioned media were examined by gelatin zymography. (b) Cells were processed for E-cadherin immunofluorescence using anti-E-cadherin ectodomain antibody (1 : 300) and Alexa Fluor 488-conjugated secondary antibody (1 : 500; green). Blue-DAPI-stained nuclei.

    Article Snippet: Cells were fixed in 4% PFA for 20 minutes at room temperature, followed by immunostaining with anti-E-cadherin (hecd-1 clone, Zymed, San Francisco, CA; 1 : 300) and Alexa-Fluor 488-conjugated secondary antibody (1 : 500).

    Techniques: Inhibition, Zymography, Immunofluorescence, Staining

    Single Sister Cell Analyses (A) Procedures to evaluate the fidelity of cell division by analyzing RNA levels in each sister cell. Anti-E-cadherin (anti-E-cad) was added to keep cells in suspension. Lists of genes and primer information are described in Table S2 and S3 , respectively. (B) A pair of sister cells before the sister cell microdissection. (C) Paired sister cells were separated by providing physical pressure on the junction between sister cells using a glass pipette. (D) Sister cells were separated by the glass pipette. (E) Each sister cell was recovered by altering the temperature of the glass pipette. After one of the sister cells was transferred to AG480F slide, Quixell system automatically brought back the pipette to the first picking position, and the other sister cell could be isolated and transferred. Scale bars, 20 μm. See also Figure S1 , Tables S1 , S2 , and S3 , and Movie S1 .

    Journal: Stem Cell Reports

    Article Title: Identifying Division Symmetry of Mouse Embryonic Stem Cells: Negative Impact of DNA Methyltransferases on Symmetric Self-Renewal

    doi: 10.1016/j.stemcr.2013.08.005

    Figure Lengend Snippet: Single Sister Cell Analyses (A) Procedures to evaluate the fidelity of cell division by analyzing RNA levels in each sister cell. Anti-E-cadherin (anti-E-cad) was added to keep cells in suspension. Lists of genes and primer information are described in Table S2 and S3 , respectively. (B) A pair of sister cells before the sister cell microdissection. (C) Paired sister cells were separated by providing physical pressure on the junction between sister cells using a glass pipette. (D) Sister cells were separated by the glass pipette. (E) Each sister cell was recovered by altering the temperature of the glass pipette. After one of the sister cells was transferred to AG480F slide, Quixell system automatically brought back the pipette to the first picking position, and the other sister cell could be isolated and transferred. Scale bars, 20 μm. See also Figure S1 , Tables S1 , S2 , and S3 , and Movie S1 .

    Article Snippet: Expression Analyses of Anti-E-Cadherin-Treated and Untreated Cells Five thousand ESCs were incubated with 4 μl of anti-E-cadherin for 90 min, and then RNA was prepared using TRIzol (Invitrogen).

    Techniques: Laser Capture Microdissection, Transferring, Isolation

    Inhibitory action of TMED10 on TGF-β signaling. A , dose-dependent inhibition of TGF-β-driven reporter activity by TMED10. HepG2 cells were transfected with (SBE) 4 ), pCH110, and the indicated plasmids at two different doses. Twenty-four hours later, the cells were stimulated with 5 ng/ml TGF-β for 18 h. Significant differences from the control in the presence of TGF-β are indicated with asterisks. B , effect of TMED10Δ(I 32 -A 80 ) on the activity of TGF-β-driven transcriptional reporter. The experiments were performed as described in A . The combined total amount of TMED10 with TMED10Δ(I 32 -A 80 ) was the same in all columns. Significant differences from the control in the presence of TGF-β are indicated with asterisks. C , inhibitory ability of TMED10 on BMP signaling. The experiments were performed as described in A except for the addition of 25 ng/ml BMP-6. Significant differences from the control in the presence of TGF-β or BMP are indicated with asterisks. D , illustration of C-terminal deletion of TMED10. SP , signal peptide; TM , transmembrane E , dispensation of the C-terminal end of TMED10 for its inhibitory action. The experiments were performed as described in A . Significant differences from the control in the presence of TGF-β are indicated with asterisks. F , inhibition of Smad2 phosphorylation by TMED10 in HaCaT cells. HaCaT cells carrying TMED10/FLAG by the method of lentiviral gene transfer were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-phosphorylated Smad2 ( PS2 ) ( top panel ), anti-Smad2 ( second panel ), anti-FLAG ( third panel ), and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses ( WB ). The expression of phosphorylated Smad2 upon TGF-β stimulation was normalized using the intensity of the band corresponding to Smad2. Relative expression was calculated relative to the value for pLV-CMV-IRES-Puro-infected cells in the absence of TGF-β. G , overexpression of TMED10/FLAG by adenoviral transfer. NMuMG cells were infected with TMED10/FLAG-expressing adenovirus. After preparation of the cell lysates, anti-FLAG ( top panel ) or anti-β-actin antibody ( bottom panel ) was used. H , extension of E-cadherin expression in NMuMG cells expressing TMED10/FLAG upon TGF-β stimulation. TMED10 were introduced into NMuMG cells by an adenoviral gene transfer system as described in G . Forty hours after infection, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-E-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of E-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. I , inhibition of N-cadherin expression by TMED10 in NMuMG cells. After gene transfer of TMED10/FLAG by adenovirus, the cells were cultured for 40 h. Subsequently, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-N-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of N-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. J , expression of TMED10 mRNA upon TGF-β stimulation. HepG2 cells were stimulated with 5 ng/ml TGF-β at the different time points. After preparation of total RNA from the cells, PCR was carried out using specific primer sets. As the positive control, the TMEPAI gene, which is well known as a TGF-β target gene, was used. Before qPCR, the amplified PCR product using each primer set could be seen in the agarose gel as a single band. Significant differences from the cells without the treatment of TGF-β are indicated with asterisks. K , overexpression of TMED10/FLAG in A549 cells by adenoviral gene transfer. The experiment was performed as described in G. L , inhibition of TGF-β-induced cell migration by TMED10. After adenoviral infection as described in K , A549 cells were seeded on the upper membrane of the Boyden chamber. Subsequently, 5 ng/ml TGF-β was added to the lower chamber for 18 h. The cells were then stained with hematoxylin/eosin solution, and the number of transmigrated cells was counted. Probability values below 0.05, 0.01, and 0.001 were considered significant: *, p

    Journal: The Journal of Biological Chemistry

    Article Title: TMED10 Protein Interferes with Transforming Growth Factor (TGF)-β Signaling by Disrupting TGF-β Receptor Complex Formation *

    doi: 10.1074/jbc.M116.769109

    Figure Lengend Snippet: Inhibitory action of TMED10 on TGF-β signaling. A , dose-dependent inhibition of TGF-β-driven reporter activity by TMED10. HepG2 cells were transfected with (SBE) 4 ), pCH110, and the indicated plasmids at two different doses. Twenty-four hours later, the cells were stimulated with 5 ng/ml TGF-β for 18 h. Significant differences from the control in the presence of TGF-β are indicated with asterisks. B , effect of TMED10Δ(I 32 -A 80 ) on the activity of TGF-β-driven transcriptional reporter. The experiments were performed as described in A . The combined total amount of TMED10 with TMED10Δ(I 32 -A 80 ) was the same in all columns. Significant differences from the control in the presence of TGF-β are indicated with asterisks. C , inhibitory ability of TMED10 on BMP signaling. The experiments were performed as described in A except for the addition of 25 ng/ml BMP-6. Significant differences from the control in the presence of TGF-β or BMP are indicated with asterisks. D , illustration of C-terminal deletion of TMED10. SP , signal peptide; TM , transmembrane E , dispensation of the C-terminal end of TMED10 for its inhibitory action. The experiments were performed as described in A . Significant differences from the control in the presence of TGF-β are indicated with asterisks. F , inhibition of Smad2 phosphorylation by TMED10 in HaCaT cells. HaCaT cells carrying TMED10/FLAG by the method of lentiviral gene transfer were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-phosphorylated Smad2 ( PS2 ) ( top panel ), anti-Smad2 ( second panel ), anti-FLAG ( third panel ), and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses ( WB ). The expression of phosphorylated Smad2 upon TGF-β stimulation was normalized using the intensity of the band corresponding to Smad2. Relative expression was calculated relative to the value for pLV-CMV-IRES-Puro-infected cells in the absence of TGF-β. G , overexpression of TMED10/FLAG by adenoviral transfer. NMuMG cells were infected with TMED10/FLAG-expressing adenovirus. After preparation of the cell lysates, anti-FLAG ( top panel ) or anti-β-actin antibody ( bottom panel ) was used. H , extension of E-cadherin expression in NMuMG cells expressing TMED10/FLAG upon TGF-β stimulation. TMED10 were introduced into NMuMG cells by an adenoviral gene transfer system as described in G . Forty hours after infection, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-E-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of E-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. I , inhibition of N-cadherin expression by TMED10 in NMuMG cells. After gene transfer of TMED10/FLAG by adenovirus, the cells were cultured for 40 h. Subsequently, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-N-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of N-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. J , expression of TMED10 mRNA upon TGF-β stimulation. HepG2 cells were stimulated with 5 ng/ml TGF-β at the different time points. After preparation of total RNA from the cells, PCR was carried out using specific primer sets. As the positive control, the TMEPAI gene, which is well known as a TGF-β target gene, was used. Before qPCR, the amplified PCR product using each primer set could be seen in the agarose gel as a single band. Significant differences from the cells without the treatment of TGF-β are indicated with asterisks. K , overexpression of TMED10/FLAG in A549 cells by adenoviral gene transfer. The experiment was performed as described in G. L , inhibition of TGF-β-induced cell migration by TMED10. After adenoviral infection as described in K , A549 cells were seeded on the upper membrane of the Boyden chamber. Subsequently, 5 ng/ml TGF-β was added to the lower chamber for 18 h. The cells were then stained with hematoxylin/eosin solution, and the number of transmigrated cells was counted. Probability values below 0.05, 0.01, and 0.001 were considered significant: *, p

    Article Snippet: For E-cadherin expression, we performed the same experiments except using the anti-E-cadherin antibody as the primary antibody and the Alexa Fluor 555-conjugated goat anti-mouse IgG antibody ( , Thermo Fisher Scientific) as the secondary antibody instead of the above combination of primary and second antibodies.

    Techniques: Inhibition, Activity Assay, Transfection, Western Blot, Expressing, Infection, Over Expression, Cell Culture, Polymerase Chain Reaction, Positive Control, Real-time Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Migration, Staining

    Localization of Flag-tagged nectin-1 and E-cadherin in MDCK-nectin-1 cells before and after a switch to medium depleted of Ca 2+ . Cells were maintained in NC medium (A to C) or switched to LC medium for 1 h (D to F) or 2 h (G to I) and then double stained with mouse anti-Flag (B, E, H, J, and K) and rat anti-E-cadherin (C, F, and I) antibodies. (A, D, and G) Phase-contrast images. (J and K) Orthogonal sections of anti-Flag-stained MDCK-nectin-1 cells maintained in NC medium (J) or in LC medium for 2 h (K). Bar, 10 μm.

    Journal: Journal of Virology

    Article Title: Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

    doi: 10.1128/JVI.76.14.7203-7208.2002

    Figure Lengend Snippet: Localization of Flag-tagged nectin-1 and E-cadherin in MDCK-nectin-1 cells before and after a switch to medium depleted of Ca 2+ . Cells were maintained in NC medium (A to C) or switched to LC medium for 1 h (D to F) or 2 h (G to I) and then double stained with mouse anti-Flag (B, E, H, J, and K) and rat anti-E-cadherin (C, F, and I) antibodies. (A, D, and G) Phase-contrast images. (J and K) Orthogonal sections of anti-Flag-stained MDCK-nectin-1 cells maintained in NC medium (J) or in LC medium for 2 h (K). Bar, 10 μm.

    Article Snippet: The primary antibodies used were mouse monoclonal anti-nectin-1 antibodies, R1.302 ( ) and CK6 and CK8 , mouse anti-Flag (M1, Sigma), and rat anti-E-cadherin (ECCD2; Zymed) antibodies.

    Techniques: Staining

    The allograft tumor exhibited histological features of collecting duct carcinoma of Bellini and immunohistochemistry staining excluded differential diagnoses ( A ) Various histological patterns were observed: solid, cribriform intermixed with independent cells in a desmoplastic stroma (hematoxylin, eosin and saffron, original magnification X100). ( B ) Tumor cells had abundant eosinophilic cytoplasm and large irregular nuclei with prominent nucleoli (hematoxylin, eosin and saffron, original magnification X200). ( C ) Immunohistochemistry with an antibody anti-PAX8, X100, showed strong intranuclear staining in tumor cells. ( D ) Immunohistochemistry with an antibody anti-E-cadherin, X100, showed strong membranous positivity of tumor cells.

    Journal: Oncotarget

    Article Title: BK virus-associated collecting duct carcinoma of the renal allograft in a kidney-pancreas allograft recipient

    doi: 10.18632/oncotarget.24552

    Figure Lengend Snippet: The allograft tumor exhibited histological features of collecting duct carcinoma of Bellini and immunohistochemistry staining excluded differential diagnoses ( A ) Various histological patterns were observed: solid, cribriform intermixed with independent cells in a desmoplastic stroma (hematoxylin, eosin and saffron, original magnification X100). ( B ) Tumor cells had abundant eosinophilic cytoplasm and large irregular nuclei with prominent nucleoli (hematoxylin, eosin and saffron, original magnification X200). ( C ) Immunohistochemistry with an antibody anti-PAX8, X100, showed strong intranuclear staining in tumor cells. ( D ) Immunohistochemistry with an antibody anti-E-cadherin, X100, showed strong membranous positivity of tumor cells.

    Article Snippet: Immunohistochemical staining was performed using the following commercially available antibodies by a Leica BOND-MAX™ autostainer (Leica Biosystems Newcastle Ltd, UK): mouse anti-BAF47/INI1 (BD Biosciences, USA; dilution 1/50), mouse anti-cytokeratin (CK) 7 (DakoCytomation, Denmark; dilution 1/800), mouse anti-CK20 (DakoCytomation, Denmark; dilution 1/100), polyclonal rabbit anti-PAX8 (Zytomed Systems Gmbh, Berlin, Germany; dilution 1/50), mouse anti-E-cadherin (InVitrogen, Carlsbad, USA; dilution 1/25), mouse anti-vimentin (DakoCytomation, Denmark; dilution 1/200), polyclonal rabbit anti-CA9 (Novus Biologicals, Littleton, USA; dilution 1/800), mouse anti-GATA3 (BioCare Medicals, USA; dilution 1/500), polyclonal rabbit anti-p504S (BioCare Medicals, USA; ready-to-use pre-diluted) and SV40 (Roche Ventana, USA; ready-to-use pre-diluted).

    Techniques: Immunohistochemistry, Staining

    Keratinocyte sheets are more stable when cultured on Fib-Mat ( A ) The morphology of keratinocyte sheets growing on dermal Fib-Mat and collagen I (Col I) substrates. Keratinocyte sheet morphology on day 4 post seeding in DSFKM (i, ii) and on day 5 after 24 hrs in DMEM/Ham’s F12 with 2% human serum (iii, iv) imaged by phase contrast microscopy. Keratinocyte sheets, day 5, stained with an anti-E-cadherin mAb (v-viii) and visualised by confocal microscopy. Shown are overview images indicating the keratinocytes have formed E-cadherin rich cell-cell junctions (v, vi). Higher magnification images revealed keratinocytes formed a monolayer on Col I (vii) but multilayered areas were common on Fib-Mat (viii). Nuclei were stained using DAPI (blue). Scale bars are 100 μm. Representative images of four replicate experiments are shown. ( B ) Keratinocytes sheets cultured on Col I (top) and Fib-Mat (bottom) substrates. Three-dimensional Z-stacked confocal images showing keratinocyte DAPI-stained nuclei as an X-Y projection, and a Z projection along the X-axis of the X-Y image delineated by the white vertical lines. The colour coding indicates the depth of the nuclei within the cell layer. Scale bars are 100 μm. ( C ) Schematics of our interpretation of the data in ( A,B ) of this figure: Col I (i), Fib-Mat (ii). ( D ) Keratinocyte sheets dissociated from the Col I and Fib-Mat substrates after treatment with dispase, and imaged by phase contrast microscopy (i, ii). Appearance of keratinocyte sheets after being subjected to 60 mechanical inversion cycles (1 cycle/sec) on a rocker, as captured by phase contrast microscopy (iii, iv). Scale bars are 500 μm. Data were consistent across three replicate experiments.

    Journal: Scientific Reports

    Article Title: In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

    doi: 10.1038/s41598-019-54793-9

    Figure Lengend Snippet: Keratinocyte sheets are more stable when cultured on Fib-Mat ( A ) The morphology of keratinocyte sheets growing on dermal Fib-Mat and collagen I (Col I) substrates. Keratinocyte sheet morphology on day 4 post seeding in DSFKM (i, ii) and on day 5 after 24 hrs in DMEM/Ham’s F12 with 2% human serum (iii, iv) imaged by phase contrast microscopy. Keratinocyte sheets, day 5, stained with an anti-E-cadherin mAb (v-viii) and visualised by confocal microscopy. Shown are overview images indicating the keratinocytes have formed E-cadherin rich cell-cell junctions (v, vi). Higher magnification images revealed keratinocytes formed a monolayer on Col I (vii) but multilayered areas were common on Fib-Mat (viii). Nuclei were stained using DAPI (blue). Scale bars are 100 μm. Representative images of four replicate experiments are shown. ( B ) Keratinocytes sheets cultured on Col I (top) and Fib-Mat (bottom) substrates. Three-dimensional Z-stacked confocal images showing keratinocyte DAPI-stained nuclei as an X-Y projection, and a Z projection along the X-axis of the X-Y image delineated by the white vertical lines. The colour coding indicates the depth of the nuclei within the cell layer. Scale bars are 100 μm. ( C ) Schematics of our interpretation of the data in ( A,B ) of this figure: Col I (i), Fib-Mat (ii). ( D ) Keratinocyte sheets dissociated from the Col I and Fib-Mat substrates after treatment with dispase, and imaged by phase contrast microscopy (i, ii). Appearance of keratinocyte sheets after being subjected to 60 mechanical inversion cycles (1 cycle/sec) on a rocker, as captured by phase contrast microscopy (iii, iv). Scale bars are 500 μm. Data were consistent across three replicate experiments.

    Article Snippet: Keratinocyte sheets were fixed, permeabilised with 0.1% Triton-X100 in PBS, blocked with BSA/1% goat serum/PBS, and stained with 20 μg/ml of anti-E Cadherin mAb, 4A2C7 (Zymed, CA, USA), anti-mouse Alexa 488 conjugated antibody and DAPI, as detailed in immunocytochemistry analysis.

    Techniques: Cell Culture, Microscopy, Staining, Confocal Microscopy, Size-exclusion Chromatography

    Cl-Amidine treatment increases E-cadherin expression in MCF10DCIS.com xenograft tumors. a PAS staining of representative mammary tissue sections of MCF10DCIS.com xenograft mice following either vehicle alone treatment (PBS) or treatment with Cl-Amidine. PAS stained sections were prepared and imaged using the bright field optics of the Axiophot inverted microscope. Scale bar = 200 μm. b Anti-E-cadherin IF staining of tissue sections from MCF10DCIS.com xenograft tumors following vehicle alone treatment (PBS) or with Cl-Amidine treatment. Scale bar = 50 μm

    Journal: BMC Cancer

    Article Title: Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration

    doi: 10.1186/s12885-017-3354-x

    Figure Lengend Snippet: Cl-Amidine treatment increases E-cadherin expression in MCF10DCIS.com xenograft tumors. a PAS staining of representative mammary tissue sections of MCF10DCIS.com xenograft mice following either vehicle alone treatment (PBS) or treatment with Cl-Amidine. PAS stained sections were prepared and imaged using the bright field optics of the Axiophot inverted microscope. Scale bar = 200 μm. b Anti-E-cadherin IF staining of tissue sections from MCF10DCIS.com xenograft tumors following vehicle alone treatment (PBS) or with Cl-Amidine treatment. Scale bar = 50 μm

    Article Snippet: Materials The following antibodies were used: anti-PAD2 (12110–1-AP, Proteintech), anti-E-cadherin (ab1518, Abcam), anti-RhoA (2117, Cell Signaling), anti-Rac1 (05–389, Millipore), anti-Cdc42 (07–1466, Millipore), anti-pan-Citrulline (07–377, Millipore; ab6464, Abcam), and anti-β-actin (ab8227, Abcam) antibodies.

    Techniques: Expressing, Staining, Mouse Assay, Inverted Microscopy

    Enhanced cell-cell adhesion is observed in PADI2 -depleted cells. a Scratch assays were performed on scrambled-shRNA and PADI2 -shRNA cells. The cells were visualized and imaged at t = 0, 12, and 24 h, using a wide-field microscope with built-in incubation chamber, to determine the extent of the wound closure. Widths of initial wounds are indicated by lines. Arrow indicates the directional movement of the migrating cells. Scale bar = 200 μm. b Images of 2 to 3 scrambled-shRNA and PADI2 -shRNA cells were taken at t = 0, 6, and 12 h. Scale bar = 50 μm. c Whole cell lysates from scrambled-shRNA and PADI2 -shRNA cells were immunoblotted with anti-E-cadherin antibody. Anti-β-actin was used as a loading control. The intensity of the band was measured using ImageJ and was normalized to scrambled-shRNA cells

    Journal: BMC Cancer

    Article Title: Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration

    doi: 10.1186/s12885-017-3354-x

    Figure Lengend Snippet: Enhanced cell-cell adhesion is observed in PADI2 -depleted cells. a Scratch assays were performed on scrambled-shRNA and PADI2 -shRNA cells. The cells were visualized and imaged at t = 0, 12, and 24 h, using a wide-field microscope with built-in incubation chamber, to determine the extent of the wound closure. Widths of initial wounds are indicated by lines. Arrow indicates the directional movement of the migrating cells. Scale bar = 200 μm. b Images of 2 to 3 scrambled-shRNA and PADI2 -shRNA cells were taken at t = 0, 6, and 12 h. Scale bar = 50 μm. c Whole cell lysates from scrambled-shRNA and PADI2 -shRNA cells were immunoblotted with anti-E-cadherin antibody. Anti-β-actin was used as a loading control. The intensity of the band was measured using ImageJ and was normalized to scrambled-shRNA cells

    Article Snippet: Materials The following antibodies were used: anti-PAD2 (12110–1-AP, Proteintech), anti-E-cadherin (ab1518, Abcam), anti-RhoA (2117, Cell Signaling), anti-Rac1 (05–389, Millipore), anti-Cdc42 (07–1466, Millipore), anti-pan-Citrulline (07–377, Millipore; ab6464, Abcam), and anti-β-actin (ab8227, Abcam) antibodies.

    Techniques: shRNA, Microscopy, Incubation

    PKD1 conserves the epithelial phenotype in normal mammary gland cells. A: NMuMG cells were either left untreated or were treated with TGFβ1 (10 ng/ml) for 48 hours. Cell morphology was photographed (bar is 200 µm) and cells were harvested and analyzed for expression of epithelial (E-cadherin, cytokeratin) and mesenchymal (N-cadherin) markers by Western blotting with anti-N-cadherin, anti-E-cadherin, or anti-cytokeratin antibodies. Staining for actin (anti-actin) served as a loading control. B: NMuMG cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Endogenous PKD1 was immunoprecipitated (anti-PKD1) and analyzed for phosphorylation at its activation loop that correlates with its activity (anti-pS738/742-PKD), or samples were control stained for total PKD1 (anti-PKD1). C: Cells were stimulated with PMA (100 nM, 10 min), EGF (50 ng/ml, 10 min), Bradykinin (0.5 µg/ml, 10 min) or left untreated. Endogenous PKD1 was immunoprecipitated and subjected to an in vitro kinase assay using PKD substrate peptide. PKD1 activity is depicted relative to PMA-activated PKD1 (maximum activation). Equal immunoprecipitation was controlled by SDS-PAGE and immunoblot (anti-PKD1). D: NMuMG cells were either transfected with control vector or with active PKD1 (PKD1.CA, PKD1.S738E.S742E). 24 hours after transfection, cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Lysates were analyzed for expression of N-cadherin, E-cadherin, expression of PKD1, or actin as a loading control. E: NMuMG cells were stably-transfected with vector control, wildtype PKD1 or kinase-dead PKD1.K612W (PKD1.KD) Cell morphology was analyzed by brightfield microscopy (bar is 200 µm). Expression of endogenous and overexpressed PKD1 was determined by Western blot analysis using an anti-PKD1 antibody. Immunoblotting for actin (anti-actin) served as loading control.

    Journal: PLoS ONE

    Article Title: Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex

    doi: 10.1371/journal.pone.0030459

    Figure Lengend Snippet: PKD1 conserves the epithelial phenotype in normal mammary gland cells. A: NMuMG cells were either left untreated or were treated with TGFβ1 (10 ng/ml) for 48 hours. Cell morphology was photographed (bar is 200 µm) and cells were harvested and analyzed for expression of epithelial (E-cadherin, cytokeratin) and mesenchymal (N-cadherin) markers by Western blotting with anti-N-cadherin, anti-E-cadherin, or anti-cytokeratin antibodies. Staining for actin (anti-actin) served as a loading control. B: NMuMG cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Endogenous PKD1 was immunoprecipitated (anti-PKD1) and analyzed for phosphorylation at its activation loop that correlates with its activity (anti-pS738/742-PKD), or samples were control stained for total PKD1 (anti-PKD1). C: Cells were stimulated with PMA (100 nM, 10 min), EGF (50 ng/ml, 10 min), Bradykinin (0.5 µg/ml, 10 min) or left untreated. Endogenous PKD1 was immunoprecipitated and subjected to an in vitro kinase assay using PKD substrate peptide. PKD1 activity is depicted relative to PMA-activated PKD1 (maximum activation). Equal immunoprecipitation was controlled by SDS-PAGE and immunoblot (anti-PKD1). D: NMuMG cells were either transfected with control vector or with active PKD1 (PKD1.CA, PKD1.S738E.S742E). 24 hours after transfection, cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Lysates were analyzed for expression of N-cadherin, E-cadherin, expression of PKD1, or actin as a loading control. E: NMuMG cells were stably-transfected with vector control, wildtype PKD1 or kinase-dead PKD1.K612W (PKD1.KD) Cell morphology was analyzed by brightfield microscopy (bar is 200 µm). Expression of endogenous and overexpressed PKD1 was determined by Western blot analysis using an anti-PKD1 antibody. Immunoblotting for actin (anti-actin) served as loading control.

    Article Snippet: Anti-GST, anti-PKD1 and anti-14-3-3 antibodies were from Santa Cruz (Santa Cruz, CA), anti-HA, anti-FLAG (M2), anti-actin, anti-nucleolin from Sigma-Aldrich (St Louis, MO), anti-E-cadherin from BD Biosciences (San Diego, CA), anti-N-cadherin from Epitomics (Burlingame, CA), anti-Snail (ab85931), anti-cytokeratin, anti-MYC and anti-GFP from Abcam (Cambridge, MA), anti-pMOTIF (PKD substrate antibody) and anti-pS744/742-PKD antibody (recognizes S738/S742-phosphorylated PKD1) from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Western Blot, Staining, Immunoprecipitation, Activation Assay, Activity Assay, In Vitro, Kinase Assay, SDS Page, Transfection, Plasmid Preparation, Stable Transfection, Microscopy

    IL-11 Expression is Correlated with Progression of Human Cancers. a Specificity of anti-IL-11 antibody used in the study. MDA-MB-231 cells were treated with control or three different siRNAs against human Il11 , and IL-11 expression in cell lysates was analyzed by Western blotting with anti-IL-11 and anti-tubulin antibodies. Results are representative of two independent experiments. b-d Adenomas (n = 10), early (n = 10) and advanced (n = 10) colorectal cancers were stained with anti-IL-11 antibody ( b ). Representative staining of respective tumors and adjacent normal tissues. Scale bars, 100 μm. Areas ( c ) and intensities ( d ) of IL-11 + signals were calculated as in the methods. Results are mean ± SE. e Tissues of advanced colorectal cancers were stained with anti-vimentin, anti-CD45, or anti-E-cadherin antibodies along with anti-IL-11 antibody (n = 3). Right panels are enlarged images of the white box in the left panels. Signaling intensities stained with the indicated antibodies on the white arrows were calculated and plotted in the right panels. Scale bars, 100 μm. Statistical significance was determined by Mann-Whitney U test ( c, d ). **p

    Journal: bioRxiv

    Article Title: Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

    doi: 10.1101/2020.01.25.919795

    Figure Lengend Snippet: IL-11 Expression is Correlated with Progression of Human Cancers. a Specificity of anti-IL-11 antibody used in the study. MDA-MB-231 cells were treated with control or three different siRNAs against human Il11 , and IL-11 expression in cell lysates was analyzed by Western blotting with anti-IL-11 and anti-tubulin antibodies. Results are representative of two independent experiments. b-d Adenomas (n = 10), early (n = 10) and advanced (n = 10) colorectal cancers were stained with anti-IL-11 antibody ( b ). Representative staining of respective tumors and adjacent normal tissues. Scale bars, 100 μm. Areas ( c ) and intensities ( d ) of IL-11 + signals were calculated as in the methods. Results are mean ± SE. e Tissues of advanced colorectal cancers were stained with anti-vimentin, anti-CD45, or anti-E-cadherin antibodies along with anti-IL-11 antibody (n = 3). Right panels are enlarged images of the white box in the left panels. Signaling intensities stained with the indicated antibodies on the white arrows were calculated and plotted in the right panels. Scale bars, 100 μm. Statistical significance was determined by Mann-Whitney U test ( c, d ). **p

    Article Snippet: The following antibodies used in this study were obtained from the indicated sources: anti-phospho-ERK (4370, CST), anti-Ki67 (ab16667, Abcam), anti-GFP (GFP-Go-Af1480 or GFP-Rb-Af2020, Frontier Institute), anti-BrdU (BU1/75, BIO-RAD), anti-IL-11 (LS-C408373, LSBio), anti-CD45 (13917, CST), anti-CD45 (IR751, Dako), anti-podoplanin (127403, BioLegend), anti-α-SMA (ab5694, Abcam), anti-collagen I (ab34710, Abcam), anti-collagen IV (ab6586, Abcam), anti-E-cadherin (560062, BD Biosciences), anti-E-cadherin (NCH-38, Dako), anti-vimentin (9856, CST), anti-phospho-STAT3 (9145, CST), anti-STAT3 (SC-482, Santa Cruz), anti-β-Actin (622102, Biolegend), and anti-tubulin (T5168, Sigma-Aldrich).

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot, Staining, MANN-WHITNEY

    Urothelium-specific and inducible expression of FGFR3b-S243C mutant in transgenic mice. ( A ) Transgene constructs. Line 1 (L1) represented a previously generated transgenic mouse line in which the 3.6-kB mouse uroplakin II (UPII) promoter drove the expression of a modified reverse tetracycline transactivator (rtTA-M2). pA, poly A signal. Line 2 (L2) was a newly generated transgenic mouse line in which the tetracycline responsive elements (TRE) drove the expression of hemagglutinin (HA)-tagged mouse FGFR3b-S243C mutant cDNA. S1, S2 and AS were oligonucleotide primers used for RT-PCR and sequencing (see panels ( B,C )). ( B ) RT-PCR and Western blotting of total RNAs and total proteins, respectively, from the double transgenic mice harboring both UPII-rtTA-M2 and TRE-FGFR3b-S243C transgenes. UI, un-induced mice (i.e., mice placed on a regular diet for 14 days); I, induced mice (i.e., mice placed on doxycycline-containing diet for 14 days). Three representative mice were shown for each condition. Note the specific detection of the FGFR3b-S243C on RNA and protein levels only in the induced mice. ( C ) Direct sequencing of the RT-PCR products from the S2/AS primer amplification showed the un-mutated codon 243 (TCC encoding serine (S)) in un-induced double transgenic mice, representing the endogenous wild-type FGFR3b; and the mutated codon 243 (TGC encoding cysteine ( C )) in induced mice, representing the transgene product. Only three codons were shown and the rest of the regions were all identical. ( D ) Double immunofluorescent staining using anti-HA and anti-E-cadherin (Ecad) showing the lack of expression of the FGFRb-S243C in un-induced mice or the strong expression of the mutant in induced mice (induction for 14 days). Also note the presence of both cytoplasmic and surface membrane staining of FGFR3b-S243C (overlapped with anti-Ecad; highlighted in dotted boxes). Magnification in ( D ), 200x.

    Journal: Scientific Reports

    Article Title: FGFR3b Extracellular Loop Mutation Lacks Tumorigenicity In Vivo but Collaborates with p53/pRB Deficiency to Induce High-grade Papillary Urothelial Carcinoma

    doi: 10.1038/srep25596

    Figure Lengend Snippet: Urothelium-specific and inducible expression of FGFR3b-S243C mutant in transgenic mice. ( A ) Transgene constructs. Line 1 (L1) represented a previously generated transgenic mouse line in which the 3.6-kB mouse uroplakin II (UPII) promoter drove the expression of a modified reverse tetracycline transactivator (rtTA-M2). pA, poly A signal. Line 2 (L2) was a newly generated transgenic mouse line in which the tetracycline responsive elements (TRE) drove the expression of hemagglutinin (HA)-tagged mouse FGFR3b-S243C mutant cDNA. S1, S2 and AS were oligonucleotide primers used for RT-PCR and sequencing (see panels ( B,C )). ( B ) RT-PCR and Western blotting of total RNAs and total proteins, respectively, from the double transgenic mice harboring both UPII-rtTA-M2 and TRE-FGFR3b-S243C transgenes. UI, un-induced mice (i.e., mice placed on a regular diet for 14 days); I, induced mice (i.e., mice placed on doxycycline-containing diet for 14 days). Three representative mice were shown for each condition. Note the specific detection of the FGFR3b-S243C on RNA and protein levels only in the induced mice. ( C ) Direct sequencing of the RT-PCR products from the S2/AS primer amplification showed the un-mutated codon 243 (TCC encoding serine (S)) in un-induced double transgenic mice, representing the endogenous wild-type FGFR3b; and the mutated codon 243 (TGC encoding cysteine ( C )) in induced mice, representing the transgene product. Only three codons were shown and the rest of the regions were all identical. ( D ) Double immunofluorescent staining using anti-HA and anti-E-cadherin (Ecad) showing the lack of expression of the FGFRb-S243C in un-induced mice or the strong expression of the mutant in induced mice (induction for 14 days). Also note the presence of both cytoplasmic and surface membrane staining of FGFR3b-S243C (overlapped with anti-Ecad; highlighted in dotted boxes). Magnification in ( D ), 200x.

    Article Snippet: The primary antibodies used in this study were anti-HA (Abcam, Inc., 1:1,000), anti-β-actin (Sigma, 1:2,000), anti-E-cadherin (SCBT Inc., 1:400), anti-p-MAPK (Thr202/Tyr204; Cell Signaling Technology, Inc., 1:2,000) anti-p-AKT (Ser473; Cell Signaling Technology, Inc., 1:200), anti-p-S6 (Ser235/Ser236; Cell Signaling Technology, Inc., 1:400) and anti-p-STAT (Tyr705; Cell Signaling Technology, Inc., 1:400).

    Techniques: Expressing, Mutagenesis, Transgenic Assay, Mouse Assay, Construct, Generated, Modification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Amplification, Staining

    TGF-β/TGFβR signaling is involved in podoplanin-induced epithelial-mesenchymal transition in UM-UC-5 cells. ( a – c ) UM-UC-5 cells were treated with or without TGF-β1 neutralizing mAb (1D11 mAb) or TGFβR inhibitors (LY2157299 or SB431542) for 2 h, followed by incubation with supernatants of UM-UC-5-platelet reactants for 48 h. Morphological and physiological changes in treated cells were examined by immunoblotting ( a ), immunofluorescence staining ( b ) and invasion assay using a matrigel-coated transwell chambers ( c ). ( a ) Cell lysates were immunoblotted with antibodies to N-cadherin, claudin-1, podoplanin (PDPN, clone D2-40) and TopoIIβ. ( b ) Cells were stained for anti-E-cadherin (green), F-actin (red; phalloidin) and nuclear DNA (blue; Hoechst 33342). Scale bars represent 50 μm. ( c ) Cells were either left untreated or treated with supernatants of UM-UC-5-platelet reactants for 48 h. Next, 5 × 10 4 UM-UC-5 cells were added to the upper chambers of matrigel-overlaid membranes. After incubation for an additional 48 h at 37 °C, cells migrating through the membranes were fixed and stained with crystal violet (lower panels; scale bars represent 200 μm). Optical density (OD) of crystal violet extracted from cells was measured at 540 nm and presented as a percentage of the OD values of control cells. All data are shown as means ± standard deviation (SD, n = 8). **P

    Journal: Scientific Reports

    Article Title: A critical role of platelet TGF-β release in podoplanin-mediated tumour invasion and metastasis

    doi: 10.1038/srep42186

    Figure Lengend Snippet: TGF-β/TGFβR signaling is involved in podoplanin-induced epithelial-mesenchymal transition in UM-UC-5 cells. ( a – c ) UM-UC-5 cells were treated with or without TGF-β1 neutralizing mAb (1D11 mAb) or TGFβR inhibitors (LY2157299 or SB431542) for 2 h, followed by incubation with supernatants of UM-UC-5-platelet reactants for 48 h. Morphological and physiological changes in treated cells were examined by immunoblotting ( a ), immunofluorescence staining ( b ) and invasion assay using a matrigel-coated transwell chambers ( c ). ( a ) Cell lysates were immunoblotted with antibodies to N-cadherin, claudin-1, podoplanin (PDPN, clone D2-40) and TopoIIβ. ( b ) Cells were stained for anti-E-cadherin (green), F-actin (red; phalloidin) and nuclear DNA (blue; Hoechst 33342). Scale bars represent 50 μm. ( c ) Cells were either left untreated or treated with supernatants of UM-UC-5-platelet reactants for 48 h. Next, 5 × 10 4 UM-UC-5 cells were added to the upper chambers of matrigel-overlaid membranes. After incubation for an additional 48 h at 37 °C, cells migrating through the membranes were fixed and stained with crystal violet (lower panels; scale bars represent 200 μm). Optical density (OD) of crystal violet extracted from cells was measured at 540 nm and presented as a percentage of the OD values of control cells. All data are shown as means ± standard deviation (SD, n = 8). **P

    Article Snippet: Immunofluorescence staining Cells plated onto coverslips were cultured for the indicated time periods, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4 for 15 min and permeabilized with 0.1% TritonX-100 in PBS for 5 min. Anti-E-cadherin (clone: HECD-1, Takara Bio, Shiga, Japan) and rhodamine- or Texas red-conjugated phalloidin (Life Technologies) were diluted in PBS containing 2% BSA as primary antibodies and cells were incubated for 90 min. Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies) was used as the secondary antibody and nuclei were stained with 1 μg/ml Hoechst 33342 (Life Technologies) for 5 min.

    Techniques: Incubation, Immunofluorescence, Staining, Invasion Assay, Standard Deviation

    Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by E-cadherin blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays anti-E-cadherin immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P

    Journal: Nature Communications

    Article Title: Mechanical cell competition kills cells via induction of lethal p53 levels

    doi: 10.1038/ncomms11373

    Figure Lengend Snippet: Contact-induced migration promotes compaction and cell competition. ( a , b ) Stills from movies of wild-type (WT) and GFP-labelled scrib KD co-cultures ( a ) or scrib KD homotypic cultures ( b ), see Supplementary Movie 4,5 . ( c , d ) Kymographs ( d ) from movies as in c . Velocities are shown before (above dashed white line) and after contact (below line). ( e ) Plot showing displacement of the line of contact between clones from movies as in c . The continuous line is the position of the front average±s.d.; n =number of contact lines averaged. ( f ) Single-cell tracking of trajectories of WT and scrib KD cells during competition. Heat-map representation shows time-resolved position of single cells. ( g ) Micrograph exemplifying cell shape change (arrows) after contact between WT and scrib KD cells. ( h ) Bar plot representing aspect ratio of WT and scrib KD cells as a function of distance from their contact point. n =50 cells of each type from three movies; error bars=s.d. ( i ) Distribution of angles between a cell's long axis and its direction of motion; n (WT)=18 cells; n ( scrib KD )=17 cells. ( j , k ) PIV analysis of images at time of contact (see Supplementary Movie 6 ) ( j ); and quantification of cell displacements ( k ) shows WT cells begin migrating (arrows) before scrib KD cells; n =10 cells for each type from three independent movies. Coloured lines=mean; shaded areas=s.d. ( l – o ) Disrupting cell junctions by E-cadherin blocking antibody and calcium removal prevents contact-induced migration ( m ), compaction ( n ) and delays competition ( o ) compared with control ( l ), see Supplementary Movie 8 ; error bars=s.e.m. ( p ) E-cadherin knockdown in WT cells ( E-cad KD ) prevents contact-induced migration. ( q ) E-cadherin knockdown in scrib KD cells ( scrib KD E-cad KD ) prevents contact-induced migration, see Supplementary Movie 9 . Right panel displays anti-E-cadherin immunofluorescence at end of movie (see Supplementary Fig. 2h,i ). Five independent repeats; n =10 events showing absence of directional migration, five were validated for E-cadherin levels and all five had WT levels. White dashed line=initial contact point; black dashed line=final contact point; yellow dashed line separates WT from scrib KD E-cad KD cells. * P

    Article Snippet: Antibodies used: anti-Scribble (1:2000, from Chris Doe lab), anti-E-Cadherin (DSHB DCAD2, 1:200).

    Techniques: Migration, Clone Assay, Single Cell Tracking, Blocking Assay, Immunofluorescence