e cadherin  (Addgene inc)

 
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  • 95
    Name:
    pENTR Cdh1 Plasmid 49776
    Description:
    Standard format Plasmid sent in bacteria as agar stab
    Catalog Number:
    49776
    Price:
    None
    Category:
    Plasmid
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    Structured Review

    Addgene inc e cadherin
    pENTR Cdh1 Plasmid 49776
    Standard format Plasmid sent in bacteria as agar stab
    https://www.bioz.com/result/e cadherin/product/Addgene inc
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    e cadherin - by Bioz Stars, 2021-01
    95/100 stars

    Images

    1) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    2) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    3) Product Images from "miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis"

    Article Title: miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159601

    miR-9’s mode of action on tumour progression through e-cadherin and SOCS5. In the presence of miR-9 (left), e-cadherin message is cleaved and suppressed, resulting in less protein production. This allows for accumulation of β-catenin (grey “L”), which can then diffuse into the nucleus, activate transcription factors and drive pro-survival, pro-proliferation transcription including c-Myc and Cyclin D1. c-Myc then initiates additional miR-9 message through a positive feedback signal. SOCS5 translation is likewise suppressed when miR-9 is overexpressed, resulting in less protein production. This allows phosphorylation and signal transduction, resulting in p-STAT transcriptional activation of pro-survival, proliferation, and invasion/mestatasis oncogenes. In the noncancerous tissue in which miR-9 expression levels are low (right panel), e-cadherin is produced and sequesters β-catenin, preventing activation of transcriptional events. Likewise, SOCS5 is produced and works to prevent phosphorylation of JAK kinase and STAT while also promoting ubiquitination, thus attenuating the JAK/STAT signaling cascade, and resulting in decreased transcription of survival, proliferation, and invasion gen es.
    Figure Legend Snippet: miR-9’s mode of action on tumour progression through e-cadherin and SOCS5. In the presence of miR-9 (left), e-cadherin message is cleaved and suppressed, resulting in less protein production. This allows for accumulation of β-catenin (grey “L”), which can then diffuse into the nucleus, activate transcription factors and drive pro-survival, pro-proliferation transcription including c-Myc and Cyclin D1. c-Myc then initiates additional miR-9 message through a positive feedback signal. SOCS5 translation is likewise suppressed when miR-9 is overexpressed, resulting in less protein production. This allows phosphorylation and signal transduction, resulting in p-STAT transcriptional activation of pro-survival, proliferation, and invasion/mestatasis oncogenes. In the noncancerous tissue in which miR-9 expression levels are low (right panel), e-cadherin is produced and sequesters β-catenin, preventing activation of transcriptional events. Likewise, SOCS5 is produced and works to prevent phosphorylation of JAK kinase and STAT while also promoting ubiquitination, thus attenuating the JAK/STAT signaling cascade, and resulting in decreased transcription of survival, proliferation, and invasion gen es.

    Techniques Used: Transduction, Activation Assay, Expressing, Produced

    4) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    5) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    6) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    7) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    8) Product Images from "E-cadherin represses anchorage-independent growth in sarcomas through both signaling and mechanical mechanisms"

    Article Title: E-cadherin represses anchorage-independent growth in sarcomas through both signaling and mechanical mechanisms

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0763

    E-cadherin inhibits spheroid formation by increased cell-cell adhesion. A. E-cadherin causes tighter clustering of cells during spheroid formation. B. A mechanical model illustrates the relationship between particle distance to adhesion. C. A mechanical model predicts E-cadherin drives down spheroid size through an increase in cell-cell adhesion. D-E. As predicted in the model, ectopic E-cadherin expression increases cell-cell adhesion in 143B ( D ) and U2OS ( E ) cells.
    Figure Legend Snippet: E-cadherin inhibits spheroid formation by increased cell-cell adhesion. A. E-cadherin causes tighter clustering of cells during spheroid formation. B. A mechanical model illustrates the relationship between particle distance to adhesion. C. A mechanical model predicts E-cadherin drives down spheroid size through an increase in cell-cell adhesion. D-E. As predicted in the model, ectopic E-cadherin expression increases cell-cell adhesion in 143B ( D ) and U2OS ( E ) cells.

    Techniques Used: Expressing

    Ectopic expression of E-cadherin in sarcoma cells inhibits phospho-CREB levels. A. A phospho-kinase array revealed that E-cadherin led to downregulation of phospho-CREB. B-C. E-cadherin-mediated phospho-CREB inhibition was verified by B. ELISAs and C. western blotting. D. Abrams canine osteosarcoma cells exhibited reduced phospho-CREB in E-cadherin over-expressing cells. E. QRT-PCR confirmed knockdown of CREB with two independent siRNAs. F. Western blotting to confirm knockdown of CREB in 143B cells. G. CREB knockdown led to a modest downregulation of 143B colony growth in soft agar.
    Figure Legend Snippet: Ectopic expression of E-cadherin in sarcoma cells inhibits phospho-CREB levels. A. A phospho-kinase array revealed that E-cadherin led to downregulation of phospho-CREB. B-C. E-cadherin-mediated phospho-CREB inhibition was verified by B. ELISAs and C. western blotting. D. Abrams canine osteosarcoma cells exhibited reduced phospho-CREB in E-cadherin over-expressing cells. E. QRT-PCR confirmed knockdown of CREB with two independent siRNAs. F. Western blotting to confirm knockdown of CREB in 143B cells. G. CREB knockdown led to a modest downregulation of 143B colony growth in soft agar.

    Techniques Used: Expressing, Inhibition, Western Blot, Quantitative RT-PCR

    TBX2 knockdown phenocopies E-cadherin-mediated CREB inhibition. A-B. TBX2 mRNA is downregulated in E-cadherin-expressing A. 143B cells and B. U2OS cells. C. CREB knockdown with two independent siRNAs had no effect on TBX2 mRNA. D-F. Conversely, TBX2 knockdown, verified in D. led to a significant reduction in CREB mRNA ( E ) and protein level ( F ).
    Figure Legend Snippet: TBX2 knockdown phenocopies E-cadherin-mediated CREB inhibition. A-B. TBX2 mRNA is downregulated in E-cadherin-expressing A. 143B cells and B. U2OS cells. C. CREB knockdown with two independent siRNAs had no effect on TBX2 mRNA. D-F. Conversely, TBX2 knockdown, verified in D. led to a significant reduction in CREB mRNA ( E ) and protein level ( F ).

    Techniques Used: Inhibition, Expressing

    E-cadherin inhibits anchorage-independent growth of sarcomas. A. Anchorage-independent growth of 143B cells expressing E-cadherin was significantly inhibited. B-C. E-cadherin expression leads to reduces spheroid size in B. 143B and C. U2OS cells.
    Figure Legend Snippet: E-cadherin inhibits anchorage-independent growth of sarcomas. A. Anchorage-independent growth of 143B cells expressing E-cadherin was significantly inhibited. B-C. E-cadherin expression leads to reduces spheroid size in B. 143B and C. U2OS cells.

    Techniques Used: Expressing

    Ectopic E-cadherin expression in sarcoma cells does not alter EMT. A. Ectopic expression of E-cadherin in 143B human osteosarcoma cells has no influence on mesenchymal markers (Snail, Slug, Twist, Zeb1, Vimentin). B. E-cadherin expression has no effect on migration of 143B cells. C. Images in B were collected every two hours and quantified using the IncuCyte Zoom system. D. E-cadherin expression does not change invasion in 143B cells. E. Using mRNA expression of CDH1 (E-cadherin), empirical probably density functions of EMT scores for CDH1-high (red) and CDH1-low (blue) TCGA sarcoma sample were constructed by interpolation of the EMT score histogram (E
    Figure Legend Snippet: Ectopic E-cadherin expression in sarcoma cells does not alter EMT. A. Ectopic expression of E-cadherin in 143B human osteosarcoma cells has no influence on mesenchymal markers (Snail, Slug, Twist, Zeb1, Vimentin). B. E-cadherin expression has no effect on migration of 143B cells. C. Images in B were collected every two hours and quantified using the IncuCyte Zoom system. D. E-cadherin expression does not change invasion in 143B cells. E. Using mRNA expression of CDH1 (E-cadherin), empirical probably density functions of EMT scores for CDH1-high (red) and CDH1-low (blue) TCGA sarcoma sample were constructed by interpolation of the EMT score histogram (E

    Techniques Used: Expressing, Migration, Construct

    E-cadherin upregulation is prognostic for improved outcomes in sarcoma. A-B. Osteosarcomas with elevated E-cadherin have better metastasis-free survival ( A ) and overall survival ( B ) as compared to tumors with low/no E-cadherin expression. C-D. Soft tissue sarcomas (STS) from The Cancer Genome Atlas with higher E-cadherin mRNA ( C ) and protein expression ( D ) have improved overall survival as compared to tumors with low or no E-cadherin.
    Figure Legend Snippet: E-cadherin upregulation is prognostic for improved outcomes in sarcoma. A-B. Osteosarcomas with elevated E-cadherin have better metastasis-free survival ( A ) and overall survival ( B ) as compared to tumors with low/no E-cadherin expression. C-D. Soft tissue sarcomas (STS) from The Cancer Genome Atlas with higher E-cadherin mRNA ( C ) and protein expression ( D ) have improved overall survival as compared to tumors with low or no E-cadherin.

    Techniques Used: Expressing

    9) Product Images from "Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer"

    Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

    Journal: Nature

    doi: 10.1038/nature18268

    Cx43 directly interacts with PCDH7, but not with E cadherin or N cadherin a, Schema illustrating split luciferase assay. Fusion constructs of PCDH7 and Cx43 were created with either the N or C terminal halves of luciferase, NLuc and CLuc, respectively. When these proteins are brought into proximity, luciferase is functionally reconstituted, producing photons of light. b, Cx43 and PCDH7 constructs fused with N-terminal and C-terminal firefly luciferase halves (N-luc) and (C-luc) were expressed in parental cell lines. The table (top) numerically identifies the cell line combinations used in the assays (bottom), and bioluminescence imaging (BLI) of a representative plate. c , Cx43 and PCDH7 western immunoblotting in cancer cells overexpressing fusion proteins. d, Quantification of BLI after co-culture of Cx43-CLuc/PCDH7-NLuc(+) cancer cells and astrocytes for 15 min. (3 independent experiments) e-g, Luciferase split assay to detect Cx43-E cadherin or Cx43-N cadherin interactions. Cell line combinations used in the assays are numerically identified in the table ( e ), and confirmed by western immunoblotting ( f ). Bioluminescence imaging (BLI) of a representative assay plate; cell line combinations are indicated numerically ( g ). (n ≥ 2 independent experiments).
    Figure Legend Snippet: Cx43 directly interacts with PCDH7, but not with E cadherin or N cadherin a, Schema illustrating split luciferase assay. Fusion constructs of PCDH7 and Cx43 were created with either the N or C terminal halves of luciferase, NLuc and CLuc, respectively. When these proteins are brought into proximity, luciferase is functionally reconstituted, producing photons of light. b, Cx43 and PCDH7 constructs fused with N-terminal and C-terminal firefly luciferase halves (N-luc) and (C-luc) were expressed in parental cell lines. The table (top) numerically identifies the cell line combinations used in the assays (bottom), and bioluminescence imaging (BLI) of a representative plate. c , Cx43 and PCDH7 western immunoblotting in cancer cells overexpressing fusion proteins. d, Quantification of BLI after co-culture of Cx43-CLuc/PCDH7-NLuc(+) cancer cells and astrocytes for 15 min. (3 independent experiments) e-g, Luciferase split assay to detect Cx43-E cadherin or Cx43-N cadherin interactions. Cell line combinations used in the assays are numerically identified in the table ( e ), and confirmed by western immunoblotting ( f ). Bioluminescence imaging (BLI) of a representative assay plate; cell line combinations are indicated numerically ( g ). (n ≥ 2 independent experiments).

    Techniques Used: Luciferase, Construct, Imaging, Western Blot, Co-Culture Assay

    10) Product Images from "Interstitial fluid pressure regulates collective invasion in engineered human breast tumors via Snail, vimentin, and E-cadherin"

    Article Title: Interstitial fluid pressure regulates collective invasion in engineered human breast tumors via Snail, vimentin, and E-cadherin

    Journal: Integrative biology : quantitative biosciences from nano to macro

    doi: 10.1039/c5ib00282f

    E-cadherin promotes invasion in response to IFP. ( A ) Frequency of invasion of YFP or Ecad-GFP-expressing aggregates under P base =P tip (YFP: n = 16; Ecad-GFP: n = 22), P base
    Figure Legend Snippet: E-cadherin promotes invasion in response to IFP. ( A ) Frequency of invasion of YFP or Ecad-GFP-expressing aggregates under P base =P tip (YFP: n = 16; Ecad-GFP: n = 22), P base

    Techniques Used: Expressing

    IFP regulates the expression of EMT markers in human breast cancer aggregates. ( A )–( D ) Relative transcript levels of vimentin (VIM), Snail (SNAI1), E-cadherin (CDH1), and keratin-8 (KRT8) in aggregates under P base =P tip ( n = 4), P base
    Figure Legend Snippet: IFP regulates the expression of EMT markers in human breast cancer aggregates. ( A )–( D ) Relative transcript levels of vimentin (VIM), Snail (SNAI1), E-cadherin (CDH1), and keratin-8 (KRT8) in aggregates under P base =P tip ( n = 4), P base

    Techniques Used: Expressing

    Cells expressing ectopic E-cadherin preferentially undergo collective invasion. ( A ) Phase-contrast image of an invasive protrusion from an Ecad-GFP aggregate under P base
    Figure Legend Snippet: Cells expressing ectopic E-cadherin preferentially undergo collective invasion. ( A ) Phase-contrast image of an invasive protrusion from an Ecad-GFP aggregate under P base

    Techniques Used: Expressing

    11) Product Images from "A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion"

    Article Title: A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13330-y

    Widespread expansion of oncogenic clones during perinatal development. a Diagram of MCAT-Crainbow mice. See also Table 1 and Supplementary Fig. 5 . b MCAT VilCre small intestine ( N = 10 mice, 3–6 weeks of age) prepared as a wholemount and confocal imaged. c Inset in “ b ” at higher magnification. d MCAT VilCre Crypts were color segmented, counted and normalized to the positional bias calculated in NCAT VilCre mice. Asterisk denotes statistical significance by one-way ANOVA (mTFP1 vs. EYFP: p = 0.003, mTFP1 vs. mKO: p = 0.016, EYFP vs. mKO = 3e–6). e Immunostaining for FLAG, V5, or HA epitopes (magenta) specific to each ßcat isoform in MCAT VilCre small intestine vibratome slices and merged with fluorescent lineage markers (mTFP1: cyan, EYFP: yellow, and mKO: orange). Arrows denote isoform expression with cognate lineage reporter (FLAG and mTFP1, V5 and EYFP, and HA and mKO). Corresponding insets depict higher magnification images. Arrowheads denote membrane-localized ßcat, whereas asterisk denotes nuclear-localized ßcat. Epitope stains (magenta) are also presented as merged and as a single-channel image with its cognate fluorescent lineage reporter (green). f HEK cells were transiently transfected with MCAT isoforms, fixed, stained, and imaged for the indicated epitope (magenta) and fluorescent reporter (green). Cells were also cotransfected with epithelial cadherin ( CDH1 ) as indicated. Arrows denote sequestration of ßcat at the plasma membrane, and the asterisk denotes nuclear ßcat. g Wnt signalling activity for each oncogene in the absence of CDH1 (solid bar) or in the presence of overexpressed CDH1 (hatched bar) ( N = 6 wells per condition and independently repeated in four experiments). TOP FLASH activity was normalized to WNT/RSPO-stimulated control cells (dashed line). Asterisk denotes statistical significance by two-way ANOVA and Bonferroni’s multiple comparisons test (cyan
    Figure Legend Snippet: Widespread expansion of oncogenic clones during perinatal development. a Diagram of MCAT-Crainbow mice. See also Table 1 and Supplementary Fig. 5 . b MCAT VilCre small intestine ( N = 10 mice, 3–6 weeks of age) prepared as a wholemount and confocal imaged. c Inset in “ b ” at higher magnification. d MCAT VilCre Crypts were color segmented, counted and normalized to the positional bias calculated in NCAT VilCre mice. Asterisk denotes statistical significance by one-way ANOVA (mTFP1 vs. EYFP: p = 0.003, mTFP1 vs. mKO: p = 0.016, EYFP vs. mKO = 3e–6). e Immunostaining for FLAG, V5, or HA epitopes (magenta) specific to each ßcat isoform in MCAT VilCre small intestine vibratome slices and merged with fluorescent lineage markers (mTFP1: cyan, EYFP: yellow, and mKO: orange). Arrows denote isoform expression with cognate lineage reporter (FLAG and mTFP1, V5 and EYFP, and HA and mKO). Corresponding insets depict higher magnification images. Arrowheads denote membrane-localized ßcat, whereas asterisk denotes nuclear-localized ßcat. Epitope stains (magenta) are also presented as merged and as a single-channel image with its cognate fluorescent lineage reporter (green). f HEK cells were transiently transfected with MCAT isoforms, fixed, stained, and imaged for the indicated epitope (magenta) and fluorescent reporter (green). Cells were also cotransfected with epithelial cadherin ( CDH1 ) as indicated. Arrows denote sequestration of ßcat at the plasma membrane, and the asterisk denotes nuclear ßcat. g Wnt signalling activity for each oncogene in the absence of CDH1 (solid bar) or in the presence of overexpressed CDH1 (hatched bar) ( N = 6 wells per condition and independently repeated in four experiments). TOP FLASH activity was normalized to WNT/RSPO-stimulated control cells (dashed line). Asterisk denotes statistical significance by two-way ANOVA and Bonferroni’s multiple comparisons test (cyan

    Techniques Used: Clone Assay, Mouse Assay, Immunostaining, Expressing, Transfection, Staining, Activity Assay

    12) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    13) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    14) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    15) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    16) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    17) Product Images from "Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin"

    Article Title: Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2013.07.012

    FadAc activates E-cadherin-mediated cellular signaling
    Figure Legend Snippet: FadAc activates E-cadherin-mediated cellular signaling

    Techniques Used:

    E-cadherin is FadA receptor
    Figure Legend Snippet: E-cadherin is FadA receptor

    Techniques Used:

    Fn adheres to and invades E-cadherin-expressing CRC cells
    Figure Legend Snippet: Fn adheres to and invades E-cadherin-expressing CRC cells

    Techniques Used: Expressing

    FadA promotes E-cadherin-mediated CRC tumor growth and induction of pro-inflammatory cytokines in xenograft mice
    Figure Legend Snippet: FadA promotes E-cadherin-mediated CRC tumor growth and induction of pro-inflammatory cytokines in xenograft mice

    Techniques Used: Mouse Assay

    Fn and FadA stimulate proliferation of human colon cancer cells via E-cadherin
    Figure Legend Snippet: Fn and FadA stimulate proliferation of human colon cancer cells via E-cadherin

    Techniques Used:

    18) Product Images from "A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion"

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    Journal: Nature cell biology

    doi: 10.1038/ncb3478

    E-cadherin is required for force transmission between CAFs and A431 cells (a) Net force transmitted between CAFs and A431 cells before the onset of contact and during contact. Experiments were performed under control conditions and after depletion of E-cadherin in the A431 cells using CRISPR/Cas9. The white bar indicates background noise levels. (-/+) n=12 CAFs from 9 independent experiments, (+/+) n=13 CAFs from 9 independent experiments, (-/-) n=13 CAFs from 2 independent experiments, (+/-) n=17 CAFs from 2 independent experiments, n=13 image regions from 8 independent experiments (noise level). Error bars represent s.e.m. *** indicates p
    Figure Legend Snippet: E-cadherin is required for force transmission between CAFs and A431 cells (a) Net force transmitted between CAFs and A431 cells before the onset of contact and during contact. Experiments were performed under control conditions and after depletion of E-cadherin in the A431 cells using CRISPR/Cas9. The white bar indicates background noise levels. (-/+) n=12 CAFs from 9 independent experiments, (+/+) n=13 CAFs from 9 independent experiments, (-/-) n=13 CAFs from 2 independent experiments, (+/-) n=17 CAFs from 2 independent experiments, n=13 image regions from 8 independent experiments (noise level). Error bars represent s.e.m. *** indicates p

    Techniques Used: Transmission Assay, CRISPR

    The E-cadherin/N-cadherin junction enables collective cancer cell invasion in 3D (a-e) Fluorescence images of spheroids containing different mixtures of CAFs and A431 cells after 60 hours of invasion in an organotypic ECM. (a) 1:1 mixture of control A431 (YPet) and control CAFs (KEIMA). (b) 1:1 mixture of A431-EcadKO (mCherry) and control CAFs (KEIMA). (c) 1:1:2 mixture of A431 control (YPet), A431-EcadKO (mCherry), and control CAFs (KEIMA). Arrow points to one A431-EcadKO cell in the invasive strand. (d) 1:1 mixture of A431 control (YPet) and CAFs-siRNA (KEIMA). (e) for additional spheroid conditions. Scale bars, 100 µm. (f) Average number of strands per spheroid in the conditions shown in (a-e) , and CAF-siCT and A431-αcatWT. Number of spheroids measured: n=24 (control), n=18 (EKO, P
    Figure Legend Snippet: The E-cadherin/N-cadherin junction enables collective cancer cell invasion in 3D (a-e) Fluorescence images of spheroids containing different mixtures of CAFs and A431 cells after 60 hours of invasion in an organotypic ECM. (a) 1:1 mixture of control A431 (YPet) and control CAFs (KEIMA). (b) 1:1 mixture of A431-EcadKO (mCherry) and control CAFs (KEIMA). (c) 1:1:2 mixture of A431 control (YPet), A431-EcadKO (mCherry), and control CAFs (KEIMA). Arrow points to one A431-EcadKO cell in the invasive strand. (d) 1:1 mixture of A431 control (YPet) and CAFs-siRNA (KEIMA). (e) for additional spheroid conditions. Scale bars, 100 µm. (f) Average number of strands per spheroid in the conditions shown in (a-e) , and CAF-siCT and A431-αcatWT. Number of spheroids measured: n=24 (control), n=18 (EKO, P

    Techniques Used: Fluorescence

    Afadin and nectins 2 and 3 are required for CAF-led migration of cancer cells and for CAF polarization. (a) Confocal images of nectin-3 (blue), N-cadherin (green), E-cadherin (red) in a co-culture of CAFs and A431 cells (upper panels); nectin-2 (blue), N-cadherin (green), E-cadherin (red) (middle panels); afadin (blue), N-cadherin (green), E-cadherin (red) (lower panels). Yellow arrows show the localization of the CAF/A431 cell contact. Images representative of 2 samples. Scale bars, 5 µm. (b) Staining of afadin (green), and p120catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell. Images representative of 5 samples. Scale bar is 10μm. (c) Fraction of “leaders” vs “loners” in CAF-siCT (n=90 CAFs) and CAF-siAF (n=95 CAFs). Data pooled from 3 independent experiments. *** indicates P
    Figure Legend Snippet: Afadin and nectins 2 and 3 are required for CAF-led migration of cancer cells and for CAF polarization. (a) Confocal images of nectin-3 (blue), N-cadherin (green), E-cadherin (red) in a co-culture of CAFs and A431 cells (upper panels); nectin-2 (blue), N-cadherin (green), E-cadherin (red) (middle panels); afadin (blue), N-cadherin (green), E-cadherin (red) (lower panels). Yellow arrows show the localization of the CAF/A431 cell contact. Images representative of 2 samples. Scale bars, 5 µm. (b) Staining of afadin (green), and p120catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell. Images representative of 5 samples. Scale bar is 10μm. (c) Fraction of “leaders” vs “loners” in CAF-siCT (n=90 CAFs) and CAF-siAF (n=95 CAFs). Data pooled from 3 independent experiments. *** indicates P

    Techniques Used: Migration, Co-Culture Assay, Staining

    CAFs and A431 cells form heterophilic E-cadherin/N-cadherin junctions (a) TEM image of contact (white arrows) between a CAF and a A431 cell. Image representative of 20 contacts from 3 independent experiments. Scale bar 100nm. (b) mRNA expression levels of E-,N- and P-cadherin in CAFs and A431 cells measured using QRT-PCR. Bars show average of technical triplicates. (c) Confocal immunofluorescence images of N-cadherin (red), E-cadherin (green), and CAGAP-mcherry (constitutively expressed by CAFs as a marker) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (d) Confocal immunofluorescence images of N-cadherin, P-cadherin, and CAGAP-mcherry (CAFs) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (e) SIM immunofluorescence images of N-cadherin (green), E-cadherin (yellow), β-catenin (red) and F-actin (blue) at a contact between CAF and A431 cell. Image representative of 15 samples. Scale bar is 1μm for zoomed areas, 10μm for merged overview projection . (f) STORM image of N-cadherin/E-cadherin localization at the contact between CAF and A431 cell. Image representative of 3 samples. Scale bar, 500nm. (g) Time-lapse images of a CAF expressing N-cadherin-GFP contacting A431 cells expressing E-cadherin-WT (red) (upper panels) or A431 cells expressing E-cadherin-W2A mutant (red) (lower panels), scale bars, 20µm . (h) Stacked histogram of life-time of the E-cadherin/N-cadherin junction (based on the E-Cadherin and N-cadherin fluorescent signals) at the contact between CAFs and A431 cells, for CAFs mixed with A431-EcadWT cells (rescue control, n=14 contacts from 3 independent experiments) and A431-EcadW2A mutant cells (n=28 contacts from 3 independent experiments). Data are pooled in three categories of contact life-time, from 0 to 30 min, from 30 to 60 min, and longer than 60 min duration. *** indicates p=0.0007, Chi-squared test.
    Figure Legend Snippet: CAFs and A431 cells form heterophilic E-cadherin/N-cadherin junctions (a) TEM image of contact (white arrows) between a CAF and a A431 cell. Image representative of 20 contacts from 3 independent experiments. Scale bar 100nm. (b) mRNA expression levels of E-,N- and P-cadherin in CAFs and A431 cells measured using QRT-PCR. Bars show average of technical triplicates. (c) Confocal immunofluorescence images of N-cadherin (red), E-cadherin (green), and CAGAP-mcherry (constitutively expressed by CAFs as a marker) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (d) Confocal immunofluorescence images of N-cadherin, P-cadherin, and CAGAP-mcherry (CAFs) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (e) SIM immunofluorescence images of N-cadherin (green), E-cadherin (yellow), β-catenin (red) and F-actin (blue) at a contact between CAF and A431 cell. Image representative of 15 samples. Scale bar is 1μm for zoomed areas, 10μm for merged overview projection . (f) STORM image of N-cadherin/E-cadherin localization at the contact between CAF and A431 cell. Image representative of 3 samples. Scale bar, 500nm. (g) Time-lapse images of a CAF expressing N-cadherin-GFP contacting A431 cells expressing E-cadherin-WT (red) (upper panels) or A431 cells expressing E-cadherin-W2A mutant (red) (lower panels), scale bars, 20µm . (h) Stacked histogram of life-time of the E-cadherin/N-cadherin junction (based on the E-Cadherin and N-cadherin fluorescent signals) at the contact between CAFs and A431 cells, for CAFs mixed with A431-EcadWT cells (rescue control, n=14 contacts from 3 independent experiments) and A431-EcadW2A mutant cells (n=28 contacts from 3 independent experiments). Data are pooled in three categories of contact life-time, from 0 to 30 min, from 30 to 60 min, and longer than 60 min duration. *** indicates p=0.0007, Chi-squared test.

    Techniques Used: Transmission Electron Microscopy, Expressing, Quantitative RT-PCR, Immunofluorescence, Marker, Co-Culture Assay, Mutagenesis

    Evidence of E-cadherin/N-cadherin junctions in lung adenocarcinoma and vulval squamous cell carcinoma (a,b) for a third patient). (c) Immunostaining of the contact between cancer cells and CAFs both isolated from one patient with vulval squamous cell carcinoma. N-cadherin (red), E-cadherin (green), F-actin (blue). Images representative of 2 patient samples. Scale bar, 5µm. (d) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431 (green), CAF (red), and collagen second harmonic (magenta), arrows highlight the different tumor components. Images representative of 3 samples. Scale bar is 20μm. (e) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431-Ecad-Ruby and vulval CAF-Ncad-GFP. White arrow highlights heterotypic contact. Images representative of 3 samples. Scale bar is 20μm. (f) Images show staining of F-actin (blue), E-cadherin (green), and αSMA (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, v - vessel. Images representative of 5 samples. Scale bar, 10μm. (g) Staining of fibronectin (magenta), active integrin β1 (green), and β-catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, yellow arrow highlights integrin/ECM contact by CAF, BM – basement membrane. Images representative of 5 samples. Scale bar, 10μm.
    Figure Legend Snippet: Evidence of E-cadherin/N-cadherin junctions in lung adenocarcinoma and vulval squamous cell carcinoma (a,b) for a third patient). (c) Immunostaining of the contact between cancer cells and CAFs both isolated from one patient with vulval squamous cell carcinoma. N-cadherin (red), E-cadherin (green), F-actin (blue). Images representative of 2 patient samples. Scale bar, 5µm. (d) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431 (green), CAF (red), and collagen second harmonic (magenta), arrows highlight the different tumor components. Images representative of 3 samples. Scale bar is 20μm. (e) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431-Ecad-Ruby and vulval CAF-Ncad-GFP. White arrow highlights heterotypic contact. Images representative of 3 samples. Scale bar is 20μm. (f) Images show staining of F-actin (blue), E-cadherin (green), and αSMA (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, v - vessel. Images representative of 5 samples. Scale bar, 10μm. (g) Staining of fibronectin (magenta), active integrin β1 (green), and β-catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, yellow arrow highlights integrin/ECM contact by CAF, BM – basement membrane. Images representative of 5 samples. Scale bar, 10μm.

    Techniques Used: Immunostaining, Isolation, Imaging, Staining

    Heterophilic E-cadherin/N-cadherin junctions withstand forces and trigger mechanotransduction (a) Illustration of the magnetic tweezers experimental setup. (b) Bead detachment data in A431 cells (CT), A431-EcadKO cells (EKO) and A431 cells pre-treated with E-cadherin blocking antibody (AbE). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to A431 cells after application of a force pulse. (c) Bead detachment data in CAFs transfected with siRNA Control (CT) and CAF-siNcad (siN). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to CAFs after application of a force pulse. (d) Illustration of the magnetic twisting experimental setup. (e) Representative fluorescence (top) and bright field (bottom) images showing the recruitment of β-catenin, P-cadherin, and E-cadherin in A431 cells subjected to magnetic stimulation using N-cadherin-coated magnetic beads. Yellow asterisks indicate the location of the beads. Scale bars, 5µm. (f) Quantification of the recruitment of β-catenin, P-cadherin and N-cadherin mediated by N-cadherin coated beads with/without (+/- Force) mechanical stimulation. (g) Representative bead traces for A431 cells and CAFs in response to a series of force pulses applied to beads coated with N-cadherin (red), E-cadherin (blue), P-cadherin (green) or uncoated (black). Vertical bars, 200nm. (h) Stiffening of the A431 cell-bead contact defined as the time evolution of the ratio between applied force and bead displacement relative to baseline (N-,E-,P-cadherin coated beads, and uncoated beads). (i) Stiffening of the CAF-bead contact. (j) Stiffening of the cell/E-cadherin-coated bead contact for control A431 cells (A431-WT) and α-catenin mutants. (k) Stiffening of the cell/N-cadherin-coated bead contact for A431-WT cells and α-catenin mutants. (l) for sample numbers and statistical analysis. Error bars represent s.e.m.
    Figure Legend Snippet: Heterophilic E-cadherin/N-cadherin junctions withstand forces and trigger mechanotransduction (a) Illustration of the magnetic tweezers experimental setup. (b) Bead detachment data in A431 cells (CT), A431-EcadKO cells (EKO) and A431 cells pre-treated with E-cadherin blocking antibody (AbE). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to A431 cells after application of a force pulse. (c) Bead detachment data in CAFs transfected with siRNA Control (CT) and CAF-siNcad (siN). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to CAFs after application of a force pulse. (d) Illustration of the magnetic twisting experimental setup. (e) Representative fluorescence (top) and bright field (bottom) images showing the recruitment of β-catenin, P-cadherin, and E-cadherin in A431 cells subjected to magnetic stimulation using N-cadherin-coated magnetic beads. Yellow asterisks indicate the location of the beads. Scale bars, 5µm. (f) Quantification of the recruitment of β-catenin, P-cadherin and N-cadherin mediated by N-cadherin coated beads with/without (+/- Force) mechanical stimulation. (g) Representative bead traces for A431 cells and CAFs in response to a series of force pulses applied to beads coated with N-cadherin (red), E-cadherin (blue), P-cadherin (green) or uncoated (black). Vertical bars, 200nm. (h) Stiffening of the A431 cell-bead contact defined as the time evolution of the ratio between applied force and bead displacement relative to baseline (N-,E-,P-cadherin coated beads, and uncoated beads). (i) Stiffening of the CAF-bead contact. (j) Stiffening of the cell/E-cadherin-coated bead contact for control A431 cells (A431-WT) and α-catenin mutants. (k) Stiffening of the cell/N-cadherin-coated bead contact for A431-WT cells and α-catenin mutants. (l) for sample numbers and statistical analysis. Error bars represent s.e.m.

    Techniques Used: Blocking Assay, Transfection, Fluorescence, Magnetic Beads

    19) Product Images from "Small-Molecule Ferroptotic Agents with Potential to Selectively Target Cancer Stem Cells"

    Article Title: Small-Molecule Ferroptotic Agents with Potential to Selectively Target Cancer Stem Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42251-5

    Modulation of ACSL4 levels by E-cadherin. Western blotting was used to measure ACSL4 in the indicated cell lines. Actin was used a loading control and the average ratio of ACSL4/Actin from 6 separate experiments is shown (4 independent lysates).
    Figure Legend Snippet: Modulation of ACSL4 levels by E-cadherin. Western blotting was used to measure ACSL4 in the indicated cell lines. Actin was used a loading control and the average ratio of ACSL4/Actin from 6 separate experiments is shown (4 independent lysates).

    Techniques Used: Western Blot

    E-Cadherin modulates 4 sensitivity. ( A ) Phase contrast and immunofluorescence imaging. E-Cadherin was re-expressed in NCI-H522 as described in the methods. Phase contrast indicates closer cell contact in E-cadherin expressing cells. ( B ) Close contact is evident by lower pixel intensities in line scans at the position of cell contact (4 cell-cell contact regions are shown). ( C ) Western analysis of E-cadherin. Cells were exposed for 14 hours with the compounds indicated before analysis. ( D ) Effects of E-cadherin on viability. Viability was determined 2 days post drug addition. ( E ) Knock-out of E-cadherin using CRISPR. The e-cadherin gene was disrupted in HCT116 colon cancer cells using CRISPR; western blot of parental and E-cadherin −/− clone is shown ( F ) Phase contrast imaging of parental and E-cadherin knockout cells. ( G ) 4 sensitivity after E-cadherin knockout. Wild-type and E-cadherin −/− HCT116 cells were exposed to 20 μM 4 for three days. Viability was measured using methylene blue staining. ( H ) Effect of salinomycin on NCI-H522 cells. Cells were exposed to the compounds indicated and viability determined 4 days later.
    Figure Legend Snippet: E-Cadherin modulates 4 sensitivity. ( A ) Phase contrast and immunofluorescence imaging. E-Cadherin was re-expressed in NCI-H522 as described in the methods. Phase contrast indicates closer cell contact in E-cadherin expressing cells. ( B ) Close contact is evident by lower pixel intensities in line scans at the position of cell contact (4 cell-cell contact regions are shown). ( C ) Western analysis of E-cadherin. Cells were exposed for 14 hours with the compounds indicated before analysis. ( D ) Effects of E-cadherin on viability. Viability was determined 2 days post drug addition. ( E ) Knock-out of E-cadherin using CRISPR. The e-cadherin gene was disrupted in HCT116 colon cancer cells using CRISPR; western blot of parental and E-cadherin −/− clone is shown ( F ) Phase contrast imaging of parental and E-cadherin knockout cells. ( G ) 4 sensitivity after E-cadherin knockout. Wild-type and E-cadherin −/− HCT116 cells were exposed to 20 μM 4 for three days. Viability was measured using methylene blue staining. ( H ) Effect of salinomycin on NCI-H522 cells. Cells were exposed to the compounds indicated and viability determined 4 days later.

    Techniques Used: Immunofluorescence, Imaging, Expressing, Western Blot, Knock-Out, CRISPR, Staining

    Mesenchymal markers correlate with compound sensitivity. ( A ) Correlation of low E-cadherin with drug sensitivity. Survival data with 1 was obtained from our own 60 cell line NCI screen. Other compounds were queried using CellMiner to obtain NCI-60 drug sensitivity data sets 53 , 54 . E-Cadherin (cdh1) expression levels were obtained from NCBI (Geo Dataset GDS4296) 55 . ( B ) Drug sensitivity correlates with high vimentin expression. Expression and survival data was collected as described in “A”.
    Figure Legend Snippet: Mesenchymal markers correlate with compound sensitivity. ( A ) Correlation of low E-cadherin with drug sensitivity. Survival data with 1 was obtained from our own 60 cell line NCI screen. Other compounds were queried using CellMiner to obtain NCI-60 drug sensitivity data sets 53 , 54 . E-Cadherin (cdh1) expression levels were obtained from NCBI (Geo Dataset GDS4296) 55 . ( B ) Drug sensitivity correlates with high vimentin expression. Expression and survival data was collected as described in “A”.

    Techniques Used: Expressing

    20) Product Images from "Dynamics of Tissue-Induced Alignment of Fibrous Extracellular Matrix"

    Article Title: Dynamics of Tissue-Induced Alignment of Fibrous Extracellular Matrix

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2017.06.046

    Disrupting the intercellular transmission of force slows matrix alignment and decreases tissue-induced strain. ( A ) Percentage of unaligned fibers over time for EpH4 mouse mammary epithelial tissues transduced with EΔ, a mutant form of E-cadherin lacking the β -catenin-binding domain ( dotted line ), or GFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( B ) Radial strain over time exerted by EpH4 mammary epithelial tissues transduced with EΔ ( dotted line ) or GFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( C ) Confocal image of an EΔ-expressing EpH4 mammary epithelial tissue at 6 h after seeding. ( D ) Percentage of unaligned fibers over time for tissues comprised of MDA-MB-231 breast cancer cells expressing Ecad-GFP ( dotted line ) or YFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( E ) Radial strain over time exerted by tissues comprised of MDA-MB-231 breast cancer cells expressing Ecad-GFP ( dotted line ) or YFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( F ) Confocal image of an Ecad-GFP-expressing MDA-MB-231 tissue at 6 h after seeding. ∗ p
    Figure Legend Snippet: Disrupting the intercellular transmission of force slows matrix alignment and decreases tissue-induced strain. ( A ) Percentage of unaligned fibers over time for EpH4 mouse mammary epithelial tissues transduced with EΔ, a mutant form of E-cadherin lacking the β -catenin-binding domain ( dotted line ), or GFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( B ) Radial strain over time exerted by EpH4 mammary epithelial tissues transduced with EΔ ( dotted line ) or GFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( C ) Confocal image of an EΔ-expressing EpH4 mammary epithelial tissue at 6 h after seeding. ( D ) Percentage of unaligned fibers over time for tissues comprised of MDA-MB-231 breast cancer cells expressing Ecad-GFP ( dotted line ) or YFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( E ) Radial strain over time exerted by tissues comprised of MDA-MB-231 breast cancer cells expressing Ecad-GFP ( dotted line ) or YFP control ( solid line ). The mean ± SE of three independent experimental replicates is shown. ( F ) Confocal image of an Ecad-GFP-expressing MDA-MB-231 tissue at 6 h after seeding. ∗ p

    Techniques Used: Transmission Assay, Transduction, Mutagenesis, Binding Assay, Expressing, Multiple Displacement Amplification

    Breast cancer cells that express low levels of E-cadherin are slower to align their surrounding matrix than non-malignant mammary epithelial cells or breast cancer cells that express higher levels of E-cadherin. ( A ) Percentage of unaligned fibers over time for tissues comprised of MDA-MB-231 human breast cancer cells. The mean ± SE of three independent experimental replicates is shown. ( B ) Radial strain over time exerted by tissues comprised of MDA-MB-231 human breast cancer cells. The mean ± SE of three independent experimental replicates is shown. ( C ) Confocal image of a representative tissue comprised of MDA-MB-231 human breast cancer cells at 6 h after seeding. ( D ) Percentage of unaligned fibers over time for tissues comprised of 4T1 murine mammary carcinoma cells. The mean ± SE of three independent experimental replicates is shown. ( E ) Radial strain over time exerted by tissues comprised of 4T1 murine mammary carcinoma cells. The mean ± SE of three independent experimental replicates is shown. ( F ) Confocal image of a representative tissue comprised of 4T1 murine mammary carcinoma cells at 6 h after seeding. The scale bar represents 50 μ m. To see this figure in color, go online.
    Figure Legend Snippet: Breast cancer cells that express low levels of E-cadherin are slower to align their surrounding matrix than non-malignant mammary epithelial cells or breast cancer cells that express higher levels of E-cadherin. ( A ) Percentage of unaligned fibers over time for tissues comprised of MDA-MB-231 human breast cancer cells. The mean ± SE of three independent experimental replicates is shown. ( B ) Radial strain over time exerted by tissues comprised of MDA-MB-231 human breast cancer cells. The mean ± SE of three independent experimental replicates is shown. ( C ) Confocal image of a representative tissue comprised of MDA-MB-231 human breast cancer cells at 6 h after seeding. ( D ) Percentage of unaligned fibers over time for tissues comprised of 4T1 murine mammary carcinoma cells. The mean ± SE of three independent experimental replicates is shown. ( E ) Radial strain over time exerted by tissues comprised of 4T1 murine mammary carcinoma cells. The mean ± SE of three independent experimental replicates is shown. ( F ) Confocal image of a representative tissue comprised of 4T1 murine mammary carcinoma cells at 6 h after seeding. The scale bar represents 50 μ m. To see this figure in color, go online.

    Techniques Used: Multiple Displacement Amplification

    21) Product Images from "Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma"

    Article Title: Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma

    Journal: Oral surgery, oral medicine, oral pathology and oral radiology

    doi: 10.1016/j.oooo.2016.05.018

    Different grades of OSCC express PCNA, Ki67, cyclin A, ΔNp63 and TGFβ1 in comparison to E-cadherin (a,e,i,m) Proteins expression in normal oral gingival mucosa (red) : PCNA, Ki67 and ΔNp63 revealed nuclear positivity staining in epithelia cells, while cyclin A expressed in both the epithelial cells and ECM. (b,c,f,g,j,k,n,o) Proteins expression in well- and moderately-differentiated OSCC (red): All of them showed a very similar pattern as they were in normal oral gingival mucosa. But their expression decreased in the cells toward the center of the tumor nest. (d,h,l,p) Proteins expression in poorly-differentiated OSCC (red): In the tumor nest, these proteins expressed only in a small portion of cancer cells (red), showing a significantly decreased expression fashion except for Ki67. (q–t) TGFβ1 expression in OSCC (red): TGFβ1 showed both nuclear and cytoplasmic staining in the epithelium as well as in the ECM (red). (at) E-cadherin expression in OSCC (green): E-cadherin expression was strictly within the epithelial membrane of the squamous layers, except in poorly-differentiated OSCC, it was reduced and discontinuous (green, d, h, l, p and t). In all slides, nuclei were stained with DAPI (blue). Scale Bar at 400μm (p) for low magnification; 100μm (p, inset) for higher magnification.
    Figure Legend Snippet: Different grades of OSCC express PCNA, Ki67, cyclin A, ΔNp63 and TGFβ1 in comparison to E-cadherin (a,e,i,m) Proteins expression in normal oral gingival mucosa (red) : PCNA, Ki67 and ΔNp63 revealed nuclear positivity staining in epithelia cells, while cyclin A expressed in both the epithelial cells and ECM. (b,c,f,g,j,k,n,o) Proteins expression in well- and moderately-differentiated OSCC (red): All of them showed a very similar pattern as they were in normal oral gingival mucosa. But their expression decreased in the cells toward the center of the tumor nest. (d,h,l,p) Proteins expression in poorly-differentiated OSCC (red): In the tumor nest, these proteins expressed only in a small portion of cancer cells (red), showing a significantly decreased expression fashion except for Ki67. (q–t) TGFβ1 expression in OSCC (red): TGFβ1 showed both nuclear and cytoplasmic staining in the epithelium as well as in the ECM (red). (at) E-cadherin expression in OSCC (green): E-cadherin expression was strictly within the epithelial membrane of the squamous layers, except in poorly-differentiated OSCC, it was reduced and discontinuous (green, d, h, l, p and t). In all slides, nuclei were stained with DAPI (blue). Scale Bar at 400μm (p) for low magnification; 100μm (p, inset) for higher magnification.

    Techniques Used: Expressing, Staining

    (A) TGFβ1 regulates PCNA, Ki67, cyclin E2, ΔNp63 and E-cadherin expression in UMSCC38 cells Column a). PCNA, Ki67, cyclin E2 and ΔNp63 revealed negative staining in untreated UMSCC38 cells. Column b). All the cells grown in 10% FBS/DMEM (positive control) were nuclear positive stained by PCNA, Ki67, cyclin E2 or ΔNp63. Column c–h). PCNA, Ki67, cyclin E2 and ΔNp63 expression in TGFβ1 treatment cells. The number of nuclei positively stained cells increased in a time and dose dependent manner, compared with untreated groups. Furthermore, as indicated by E-cadherin staining along the cell membrane (red), the cobblestone morphology was preserved in all treated UMSCC38 cells. (B) TGFβ1 regulates PCNA, Ki67, cyclin E2, ΔNp63 and E-cadherin expression in UMSCC11B cells. Column a–b). PCNA, Ki67, cyclin E2 and ΔNp63 expressed very similarly as that in UMSCC38 untreated and positive control cells. Column c–e). 24 h TGFβ1 treatment increased the number and intensity of PCNA, Ki67, cyclin E2 and ΔNp63 (green) expression in UMSCC11B cells compared to untreated control groups. Also the expression pattern of the epithelial marker, E-cadherin (red), was concentrated on the cell membrane, forming a continuous membranous, similar as that in the untreated cells. Column f–h). When treated with TGFβ1 for 48 h, the number of positive cells decreased significantly, and the membranous expression of E-cadherin (red) was reduced and interrupted thereafter. (C). Percentage of immunofluorescence positively stained UMSCC38 and UMSCC11B cells. For both UMSCC38 and 11B cells, only very limited untreated cells express PCNA, Ki67, cyclin E2 or ΔNp63. (a–d) Percentage of positively stained UMSCC38 cells for PCNA (a), ΔNp63 (b), Ki67 (c) and cyclin E2 (d). Over 90.0 percent of UMSCC38 cells grown in 10% FBS were positively stained for these proteins. When cells were treated with TGFβ1 for 24 h, the percentages increased as the dilutions of TGFβ1 were raised. At 48 h, the percentages raised to very similar level with that of positive control (10% FBS) groups. (e–h) Percentage of positively stained UMSCC11B cells for PCNA (e), ΔNp63 (f), Ki67 (g) and cyclin E2 (h). Almost 95.0 percent of cells grown in 10% FBS were positive stained for PCNA, ΔNp63, Ki67 and cyclin E2. When cells were treated with TGFβ1 for 24 h, the percentages increased obviously as the dilutions of TGFβ1 were raised. But at 48 h, the percentages of positively stained cells reduced obviously, very similar as that of untreated cells.
    Figure Legend Snippet: (A) TGFβ1 regulates PCNA, Ki67, cyclin E2, ΔNp63 and E-cadherin expression in UMSCC38 cells Column a). PCNA, Ki67, cyclin E2 and ΔNp63 revealed negative staining in untreated UMSCC38 cells. Column b). All the cells grown in 10% FBS/DMEM (positive control) were nuclear positive stained by PCNA, Ki67, cyclin E2 or ΔNp63. Column c–h). PCNA, Ki67, cyclin E2 and ΔNp63 expression in TGFβ1 treatment cells. The number of nuclei positively stained cells increased in a time and dose dependent manner, compared with untreated groups. Furthermore, as indicated by E-cadherin staining along the cell membrane (red), the cobblestone morphology was preserved in all treated UMSCC38 cells. (B) TGFβ1 regulates PCNA, Ki67, cyclin E2, ΔNp63 and E-cadherin expression in UMSCC11B cells. Column a–b). PCNA, Ki67, cyclin E2 and ΔNp63 expressed very similarly as that in UMSCC38 untreated and positive control cells. Column c–e). 24 h TGFβ1 treatment increased the number and intensity of PCNA, Ki67, cyclin E2 and ΔNp63 (green) expression in UMSCC11B cells compared to untreated control groups. Also the expression pattern of the epithelial marker, E-cadherin (red), was concentrated on the cell membrane, forming a continuous membranous, similar as that in the untreated cells. Column f–h). When treated with TGFβ1 for 48 h, the number of positive cells decreased significantly, and the membranous expression of E-cadherin (red) was reduced and interrupted thereafter. (C). Percentage of immunofluorescence positively stained UMSCC38 and UMSCC11B cells. For both UMSCC38 and 11B cells, only very limited untreated cells express PCNA, Ki67, cyclin E2 or ΔNp63. (a–d) Percentage of positively stained UMSCC38 cells for PCNA (a), ΔNp63 (b), Ki67 (c) and cyclin E2 (d). Over 90.0 percent of UMSCC38 cells grown in 10% FBS were positively stained for these proteins. When cells were treated with TGFβ1 for 24 h, the percentages increased as the dilutions of TGFβ1 were raised. At 48 h, the percentages raised to very similar level with that of positive control (10% FBS) groups. (e–h) Percentage of positively stained UMSCC11B cells for PCNA (e), ΔNp63 (f), Ki67 (g) and cyclin E2 (h). Almost 95.0 percent of cells grown in 10% FBS were positive stained for PCNA, ΔNp63, Ki67 and cyclin E2. When cells were treated with TGFβ1 for 24 h, the percentages increased obviously as the dilutions of TGFβ1 were raised. But at 48 h, the percentages of positively stained cells reduced obviously, very similar as that of untreated cells.

    Techniques Used: Expressing, Negative Staining, Positive Control, Staining, Marker, Immunofluorescence

    22) Product Images from "Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells"

    Article Title: Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201704102

    Loss of E-cadherin promotes efficient centrosome clustering in nontransformed cell lines. (A) Western blot analysis of E-cadherin and HSET levels in a panel of nontransformed cell lines. (B) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells after siRNA depletion of E-cadherin. (C) Quantification of centrosome clustering in cytokinesis upon DCB treatment in E-cadherin depleted cells ( n = 150). (D) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells upon CRISPR-Cas9 knockout of E-cadherin ( CDH1 −/− ; five knockout clones combined for each cell line). (E) Immunofluorescence images of control and CDH1 −/− MCF10A and HaCaT cells stained for E-cadherin (green) and DNA (blue). (F) Quantification of centrosome clustering in cytokinesis in control and CDH1 −/− cells ( n = 150). (G) Immunofluorescence images in RPE-1 cells expressing WT E-cadherin and E-cadherin DN. Cells were stained for E-cadherin (red) and DNA (blue). White arrow highlights the cell–cell junctions. (H) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN ( n = 150). (I) Analyses of the survival curves in control and CDH1 −/− MCF10A and HaCaT cells upon induction of centrosome amplification via PLK4 overexpression (PLK4 OE). For all graphics, error bars represent mean ± SD from three independent experiments. **, P
    Figure Legend Snippet: Loss of E-cadherin promotes efficient centrosome clustering in nontransformed cell lines. (A) Western blot analysis of E-cadherin and HSET levels in a panel of nontransformed cell lines. (B) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells after siRNA depletion of E-cadherin. (C) Quantification of centrosome clustering in cytokinesis upon DCB treatment in E-cadherin depleted cells ( n = 150). (D) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells upon CRISPR-Cas9 knockout of E-cadherin ( CDH1 −/− ; five knockout clones combined for each cell line). (E) Immunofluorescence images of control and CDH1 −/− MCF10A and HaCaT cells stained for E-cadherin (green) and DNA (blue). (F) Quantification of centrosome clustering in cytokinesis in control and CDH1 −/− cells ( n = 150). (G) Immunofluorescence images in RPE-1 cells expressing WT E-cadherin and E-cadherin DN. Cells were stained for E-cadherin (red) and DNA (blue). White arrow highlights the cell–cell junctions. (H) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN ( n = 150). (I) Analyses of the survival curves in control and CDH1 −/− MCF10A and HaCaT cells upon induction of centrosome amplification via PLK4 overexpression (PLK4 OE). For all graphics, error bars represent mean ± SD from three independent experiments. **, P

    Techniques Used: Western Blot, CRISPR, Knock-Out, Clone Assay, Immunofluorescence, Staining, Expressing, Amplification, Over Expression

    Loss of E-cadherin and DDR1 correlates with high levels of centrosome amplification in breast cancer. (A) Quantification of centrosome numbers in a panel of breast cancer cell lines. (B) Western blot analysis of E-cadherin, DDR1, and HSET expression in breast cancer cell lines. Red asterisk marks the cell lines with high levels of centrosome amplification. (C) Quantification of centrosome clustering in metaphase and cytokinesis in cells with high levels of centrosome amplification. Error bars represent mean ± SD from three independent experiments.
    Figure Legend Snippet: Loss of E-cadherin and DDR1 correlates with high levels of centrosome amplification in breast cancer. (A) Quantification of centrosome numbers in a panel of breast cancer cell lines. (B) Western blot analysis of E-cadherin, DDR1, and HSET expression in breast cancer cell lines. Red asterisk marks the cell lines with high levels of centrosome amplification. (C) Quantification of centrosome clustering in metaphase and cytokinesis in cells with high levels of centrosome amplification. Error bars represent mean ± SD from three independent experiments.

    Techniques Used: Amplification, Western Blot, Expressing

    Inhibition of cortical contractility downstream of E-cadherin/DDR1 complex inhibits centrosome movement. (A) Representation of the angles measured between the two closest poles in tripolar metaphases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (B) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon blebbistatin treatment (50 µM, 4 h; n = 150). Dashed line represents the mean angle distribution. (C) Quantification of centrosome clustering in cytokinesis upon depletion of HSET by siRNA (48 h). (D) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon HSET siRNA. Dashed line represents the mean angle distribution. (E) Immunofluorescence images of mitotic cells with high levels of extra centrosomes. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (F) Quantification of number of centrioles per mitotic cell in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h ( n = 150). (G) Quantification centrosome clustering in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P
    Figure Legend Snippet: Inhibition of cortical contractility downstream of E-cadherin/DDR1 complex inhibits centrosome movement. (A) Representation of the angles measured between the two closest poles in tripolar metaphases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (B) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon blebbistatin treatment (50 µM, 4 h; n = 150). Dashed line represents the mean angle distribution. (C) Quantification of centrosome clustering in cytokinesis upon depletion of HSET by siRNA (48 h). (D) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon HSET siRNA. Dashed line represents the mean angle distribution. (E) Immunofluorescence images of mitotic cells with high levels of extra centrosomes. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (F) Quantification of number of centrioles per mitotic cell in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h ( n = 150). (G) Quantification centrosome clustering in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P

    Techniques Used: Inhibition, Staining, Immunofluorescence

    Cortical contractility facilitates centrosome clustering in cells that do not express E-cadherin. (A, top) Schematic representation of AFM experiment. (Bottom) Bright-field images of chosen metaphase cells used for the stiffness measurements. Cantilever can also be observed in these images. Bar, 20 µM. (B) Quantification of apparent elasticity (Pa) in metaphase cells treated with blebbistatin (50 µM, 4 h) and calyculin A (1 µM, 2 h). (C) Quantification of apparent elasticity (Pa) in metaphase cells within a monolayer. (D) Immunofluorescence images depicting examples of bipolar clustered and multipolar telophases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µM. (Inset) High magnification of centrioles. Bar, 1 µM. (E) Quantification of centrosome clustering in telophase upon blebbistatin treatment (50 µM, 4 h; n = 150). (F) Quantification of centrosome clustering in cytokinesis upon treatment with calyculin A (1 µM, 2 h; n = 150). For all graphics, error bars represent mean ± SD from three independent experiments. *, P
    Figure Legend Snippet: Cortical contractility facilitates centrosome clustering in cells that do not express E-cadherin. (A, top) Schematic representation of AFM experiment. (Bottom) Bright-field images of chosen metaphase cells used for the stiffness measurements. Cantilever can also be observed in these images. Bar, 20 µM. (B) Quantification of apparent elasticity (Pa) in metaphase cells treated with blebbistatin (50 µM, 4 h) and calyculin A (1 µM, 2 h). (C) Quantification of apparent elasticity (Pa) in metaphase cells within a monolayer. (D) Immunofluorescence images depicting examples of bipolar clustered and multipolar telophases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µM. (Inset) High magnification of centrioles. Bar, 1 µM. (E) Quantification of centrosome clustering in telophase upon blebbistatin treatment (50 µM, 4 h; n = 150). (F) Quantification of centrosome clustering in cytokinesis upon treatment with calyculin A (1 µM, 2 h; n = 150). For all graphics, error bars represent mean ± SD from three independent experiments. *, P

    Techniques Used: Immunofluorescence, Staining

    Cortical localization of DDR1 in cells expressing E-cadherin prevents efficient centrosome clustering. (A) Immunofluorescence images showing cortical localization of DDR1 during mitosis. Cells were stained for F-actin (phalloidin, red), DDR1 (green), and DNA (blue). White arrows represent areas where there are no cell–cell contacts. (B, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion of DDR1 in MCF10A cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (C, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion DDR1 in HaCaT cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (D) Western blot analysis of the levels of E-cadherin and DDR1 in RPE-1 cells expressing exogenous WT E-cadherin and E-cadherin DN. (E) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN before and after DDR1 depletion by siRNA ( n = 150). (F) Quantification of centrosome clustering in metaphase and cytokinesis in MCF10A cells treated with 15 µM DDR1 inhibitor for 3 h. (G) Western blot analysis of RhoE levels in HaCaT cells after siRNA depletion of RhoE. (H) Quantification of centrosome clustering in cytokinesis upon RhoE depletion ( n = 150). (I) Schematic representation of RhoE-mediated regulation of cortical contractility downstream of E-cadherin. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P
    Figure Legend Snippet: Cortical localization of DDR1 in cells expressing E-cadherin prevents efficient centrosome clustering. (A) Immunofluorescence images showing cortical localization of DDR1 during mitosis. Cells were stained for F-actin (phalloidin, red), DDR1 (green), and DNA (blue). White arrows represent areas where there are no cell–cell contacts. (B, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion of DDR1 in MCF10A cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (C, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion DDR1 in HaCaT cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (D) Western blot analysis of the levels of E-cadherin and DDR1 in RPE-1 cells expressing exogenous WT E-cadherin and E-cadherin DN. (E) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN before and after DDR1 depletion by siRNA ( n = 150). (F) Quantification of centrosome clustering in metaphase and cytokinesis in MCF10A cells treated with 15 µM DDR1 inhibitor for 3 h. (G) Western blot analysis of RhoE levels in HaCaT cells after siRNA depletion of RhoE. (H) Quantification of centrosome clustering in cytokinesis upon RhoE depletion ( n = 150). (I) Schematic representation of RhoE-mediated regulation of cortical contractility downstream of E-cadherin. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    23) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    24) Product Images from "E-cadherin re-expression shows in vivo evidence for mesenchymal to epithelial transition in clonal metastatic breast tumor cells"

    Article Title: E-cadherin re-expression shows in vivo evidence for mesenchymal to epithelial transition in clonal metastatic breast tumor cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9715

    Metastasized clonal epithelial tumor cells re-express E-cadherin ( A ) A representation of firefly luciferase bioluminescent signal in organs of mice injected into the MFP with 2 × 10 6 GFP + E2 cells expressing firefly luciferase or 1 × 10 6 GFP + E2 cells expressing firefly luciferase mixed with 1 × 10 6 M1 cells. ( B ) Membrane E-cadherin expression on GFP + flow cytometry-gated cells recovered from metastatic organs. ( C ) The graph showing the difference in E-cadherin expression in E2 cells harvested from the MFP (shown in Figure 4D ) and E2 cells harvested from the corresponding metastasis (Figure 5B ). E2 is the parental cell line control.
    Figure Legend Snippet: Metastasized clonal epithelial tumor cells re-express E-cadherin ( A ) A representation of firefly luciferase bioluminescent signal in organs of mice injected into the MFP with 2 × 10 6 GFP + E2 cells expressing firefly luciferase or 1 × 10 6 GFP + E2 cells expressing firefly luciferase mixed with 1 × 10 6 M1 cells. ( B ) Membrane E-cadherin expression on GFP + flow cytometry-gated cells recovered from metastatic organs. ( C ) The graph showing the difference in E-cadherin expression in E2 cells harvested from the MFP (shown in Figure 4D ) and E2 cells harvested from the corresponding metastasis (Figure 5B ). E2 is the parental cell line control.

    Techniques Used: Luciferase, Mouse Assay, Injection, Expressing, Flow Cytometry, Cytometry

    Epithelial and mesenchymal-like clonal cell lines are tumor-derived ( A ) Flow cytometry histograms showing membrane rat neu protein expression. Red histogram = isotype control and blue histogram = neu expression. ( B ) Flow cytometric mean fluorescence intensity (MFI) values of membrane rat neu protein expression (panel 1). The MFI value was obtained by subtracting the MFI of the isotype control from the experimental value. Real-time quantitative PCR (qPCR) of cell line or tissue cDNA using rat neu specific primers (panel 2). For flow cytometry and qPCR, error bars represent repeats of 3 separate experiments. ( C ) Flow cytometric analysis of membrane E-cadherin protein expression. Red histogram = isotype control and blue histogram = E-cadherin expression. ( D ) qPCR showing relative differences in transcript expression of the epithelial marker, E-cadherin, and mesenchymal markers, vimentin, Zeb1 and Twist1 in E1, E2, M1 and M2 cell lines. NIH3T3 cells were analyzed as a mesenchymal cell line control. NA = no amplification signal. Error bars represent the average ΔΔCq from triplicate wells in 3 separate experiments. Statistical analysis was done using ordinary one-way ANOVA with a Tukey multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.
    Figure Legend Snippet: Epithelial and mesenchymal-like clonal cell lines are tumor-derived ( A ) Flow cytometry histograms showing membrane rat neu protein expression. Red histogram = isotype control and blue histogram = neu expression. ( B ) Flow cytometric mean fluorescence intensity (MFI) values of membrane rat neu protein expression (panel 1). The MFI value was obtained by subtracting the MFI of the isotype control from the experimental value. Real-time quantitative PCR (qPCR) of cell line or tissue cDNA using rat neu specific primers (panel 2). For flow cytometry and qPCR, error bars represent repeats of 3 separate experiments. ( C ) Flow cytometric analysis of membrane E-cadherin protein expression. Red histogram = isotype control and blue histogram = E-cadherin expression. ( D ) qPCR showing relative differences in transcript expression of the epithelial marker, E-cadherin, and mesenchymal markers, vimentin, Zeb1 and Twist1 in E1, E2, M1 and M2 cell lines. NIH3T3 cells were analyzed as a mesenchymal cell line control. NA = no amplification signal. Error bars represent the average ΔΔCq from triplicate wells in 3 separate experiments. Statistical analysis was done using ordinary one-way ANOVA with a Tukey multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Real-time Polymerase Chain Reaction, Marker, Amplification

    Orthotopically implanted clonal epithelial tumor cells lose E-cadherin expression over time ( A ) A flow cytometric representation of membrane E-cadherin expression in neu + gated E2 tumor cells harvested from MFP tumors at 50 mm 2 ( n = 3, day 23) and 100 mm 2 in size ( n = 5, day 61). E2 is the parental cell line control. ( B ) Graph of combined flow cytometric data shown in A. Statistical analysis was done using ordinary one-way ANOVA with a Tukey multiple comparison test. ** p ≤ 0.01, and *** p ≤ 0.001. ( C ) Flow cytometry showing the neu + cell gate from which cells in A were analyzed. ( D ) Membrane E-cadherin expression on E2 GFP + sorted cells harvested from MFP tumors (~250 mm 2 , day ~200). Mice were inoculated with 2 × 10 6 GFP + E2 cells (tumors 1 and 2) or a mix of 1 × 10 6 GFP + E2 and 1 × 10 6 M1 tumor cells (tumors 3 and 4). MFP = mammary fat pad.
    Figure Legend Snippet: Orthotopically implanted clonal epithelial tumor cells lose E-cadherin expression over time ( A ) A flow cytometric representation of membrane E-cadherin expression in neu + gated E2 tumor cells harvested from MFP tumors at 50 mm 2 ( n = 3, day 23) and 100 mm 2 in size ( n = 5, day 61). E2 is the parental cell line control. ( B ) Graph of combined flow cytometric data shown in A. Statistical analysis was done using ordinary one-way ANOVA with a Tukey multiple comparison test. ** p ≤ 0.01, and *** p ≤ 0.001. ( C ) Flow cytometry showing the neu + cell gate from which cells in A were analyzed. ( D ) Membrane E-cadherin expression on E2 GFP + sorted cells harvested from MFP tumors (~250 mm 2 , day ~200). Mice were inoculated with 2 × 10 6 GFP + E2 cells (tumors 1 and 2) or a mix of 1 × 10 6 GFP + E2 and 1 × 10 6 M1 tumor cells (tumors 3 and 4). MFP = mammary fat pad.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Mouse Assay

    E-cadherin expression in M1 cells does not rescue tumorigenesis ( A ) Stable expression of E-cadherin in M1 cells transfected with Addgene plasmid #18804 expressing murine E-cadherin. Red line = E-cadherin expression in parental M1 cell line and blue line = membrane E-cadherin expression in transfected M1 cells. ( B ) Renilla luciferase bioluminescent signal in the MFP immediately (day 0) after injection of 2 × 10 6 M1 cells expressing E-cadherin and 27 days later. The figure represents data obtained from 9 mice.
    Figure Legend Snippet: E-cadherin expression in M1 cells does not rescue tumorigenesis ( A ) Stable expression of E-cadherin in M1 cells transfected with Addgene plasmid #18804 expressing murine E-cadherin. Red line = E-cadherin expression in parental M1 cell line and blue line = membrane E-cadherin expression in transfected M1 cells. ( B ) Renilla luciferase bioluminescent signal in the MFP immediately (day 0) after injection of 2 × 10 6 M1 cells expressing E-cadherin and 27 days later. The figure represents data obtained from 9 mice.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Injection, Mouse Assay

    25) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    26) Product Images from "Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells"

    Article Title: Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201704102

    Loss of E-cadherin promotes efficient centrosome clustering in nontransformed cell lines. (A) Western blot analysis of E-cadherin and HSET levels in a panel of nontransformed cell lines. (B) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells after siRNA depletion of E-cadherin. (C) Quantification of centrosome clustering in cytokinesis upon DCB treatment in E-cadherin depleted cells ( n = 150). (D) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells upon CRISPR-Cas9 knockout of E-cadherin ( CDH1 −/− ; five knockout clones combined for each cell line). (E) Immunofluorescence images of control and CDH1 −/− MCF10A and HaCaT cells stained for E-cadherin (green) and DNA (blue). (F) Quantification of centrosome clustering in cytokinesis in control and CDH1 −/− cells ( n = 150). (G) Immunofluorescence images in RPE-1 cells expressing WT E-cadherin and E-cadherin DN. Cells were stained for E-cadherin (red) and DNA (blue). White arrow highlights the cell–cell junctions. (H) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN ( n = 150). (I) Analyses of the survival curves in control and CDH1 −/− MCF10A and HaCaT cells upon induction of centrosome amplification via PLK4 overexpression (PLK4 OE). For all graphics, error bars represent mean ± SD from three independent experiments. **, P
    Figure Legend Snippet: Loss of E-cadherin promotes efficient centrosome clustering in nontransformed cell lines. (A) Western blot analysis of E-cadherin and HSET levels in a panel of nontransformed cell lines. (B) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells after siRNA depletion of E-cadherin. (C) Quantification of centrosome clustering in cytokinesis upon DCB treatment in E-cadherin depleted cells ( n = 150). (D) Western blot analysis of E-cadherin levels in MCF10A and HaCaT cells upon CRISPR-Cas9 knockout of E-cadherin ( CDH1 −/− ; five knockout clones combined for each cell line). (E) Immunofluorescence images of control and CDH1 −/− MCF10A and HaCaT cells stained for E-cadherin (green) and DNA (blue). (F) Quantification of centrosome clustering in cytokinesis in control and CDH1 −/− cells ( n = 150). (G) Immunofluorescence images in RPE-1 cells expressing WT E-cadherin and E-cadherin DN. Cells were stained for E-cadherin (red) and DNA (blue). White arrow highlights the cell–cell junctions. (H) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN ( n = 150). (I) Analyses of the survival curves in control and CDH1 −/− MCF10A and HaCaT cells upon induction of centrosome amplification via PLK4 overexpression (PLK4 OE). For all graphics, error bars represent mean ± SD from three independent experiments. **, P

    Techniques Used: Western Blot, CRISPR, Knock-Out, Clone Assay, Immunofluorescence, Staining, Expressing, Amplification, Over Expression

    Loss of E-cadherin and DDR1 correlates with high levels of centrosome amplification in breast cancer. (A) Quantification of centrosome numbers in a panel of breast cancer cell lines. (B) Western blot analysis of E-cadherin, DDR1, and HSET expression in breast cancer cell lines. Red asterisk marks the cell lines with high levels of centrosome amplification. (C) Quantification of centrosome clustering in metaphase and cytokinesis in cells with high levels of centrosome amplification. Error bars represent mean ± SD from three independent experiments.
    Figure Legend Snippet: Loss of E-cadherin and DDR1 correlates with high levels of centrosome amplification in breast cancer. (A) Quantification of centrosome numbers in a panel of breast cancer cell lines. (B) Western blot analysis of E-cadherin, DDR1, and HSET expression in breast cancer cell lines. Red asterisk marks the cell lines with high levels of centrosome amplification. (C) Quantification of centrosome clustering in metaphase and cytokinesis in cells with high levels of centrosome amplification. Error bars represent mean ± SD from three independent experiments.

    Techniques Used: Amplification, Western Blot, Expressing

    Inhibition of cortical contractility downstream of E-cadherin/DDR1 complex inhibits centrosome movement. (A) Representation of the angles measured between the two closest poles in tripolar metaphases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (B) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon blebbistatin treatment (50 µM, 4 h; n = 150). Dashed line represents the mean angle distribution. (C) Quantification of centrosome clustering in cytokinesis upon depletion of HSET by siRNA (48 h). (D) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon HSET siRNA. Dashed line represents the mean angle distribution. (E) Immunofluorescence images of mitotic cells with high levels of extra centrosomes. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (F) Quantification of number of centrioles per mitotic cell in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h ( n = 150). (G) Quantification centrosome clustering in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P
    Figure Legend Snippet: Inhibition of cortical contractility downstream of E-cadherin/DDR1 complex inhibits centrosome movement. (A) Representation of the angles measured between the two closest poles in tripolar metaphases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (B) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon blebbistatin treatment (50 µM, 4 h; n = 150). Dashed line represents the mean angle distribution. (C) Quantification of centrosome clustering in cytokinesis upon depletion of HSET by siRNA (48 h). (D) Rose plot showing the frequency of the angles measured in MCF10A and HaCaT cells (control and CDH1 −/− ) upon HSET siRNA. Dashed line represents the mean angle distribution. (E) Immunofluorescence images of mitotic cells with high levels of extra centrosomes. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µm. (F) Quantification of number of centrioles per mitotic cell in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h ( n = 150). (G) Quantification centrosome clustering in cells overexpressing PLK4 treated or not with SAS-6 siRNA for 48 h. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P

    Techniques Used: Inhibition, Staining, Immunofluorescence

    Cortical contractility facilitates centrosome clustering in cells that do not express E-cadherin. (A, top) Schematic representation of AFM experiment. (Bottom) Bright-field images of chosen metaphase cells used for the stiffness measurements. Cantilever can also be observed in these images. Bar, 20 µM. (B) Quantification of apparent elasticity (Pa) in metaphase cells treated with blebbistatin (50 µM, 4 h) and calyculin A (1 µM, 2 h). (C) Quantification of apparent elasticity (Pa) in metaphase cells within a monolayer. (D) Immunofluorescence images depicting examples of bipolar clustered and multipolar telophases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µM. (Inset) High magnification of centrioles. Bar, 1 µM. (E) Quantification of centrosome clustering in telophase upon blebbistatin treatment (50 µM, 4 h; n = 150). (F) Quantification of centrosome clustering in cytokinesis upon treatment with calyculin A (1 µM, 2 h; n = 150). For all graphics, error bars represent mean ± SD from three independent experiments. *, P
    Figure Legend Snippet: Cortical contractility facilitates centrosome clustering in cells that do not express E-cadherin. (A, top) Schematic representation of AFM experiment. (Bottom) Bright-field images of chosen metaphase cells used for the stiffness measurements. Cantilever can also be observed in these images. Bar, 20 µM. (B) Quantification of apparent elasticity (Pa) in metaphase cells treated with blebbistatin (50 µM, 4 h) and calyculin A (1 µM, 2 h). (C) Quantification of apparent elasticity (Pa) in metaphase cells within a monolayer. (D) Immunofluorescence images depicting examples of bipolar clustered and multipolar telophases. Cells were stained for microtubules (α-Tub, red), centrioles (centrin2, green), and DNA (blue). Bar, 10 µM. (Inset) High magnification of centrioles. Bar, 1 µM. (E) Quantification of centrosome clustering in telophase upon blebbistatin treatment (50 µM, 4 h; n = 150). (F) Quantification of centrosome clustering in cytokinesis upon treatment with calyculin A (1 µM, 2 h; n = 150). For all graphics, error bars represent mean ± SD from three independent experiments. *, P

    Techniques Used: Immunofluorescence, Staining

    Cortical localization of DDR1 in cells expressing E-cadherin prevents efficient centrosome clustering. (A) Immunofluorescence images showing cortical localization of DDR1 during mitosis. Cells were stained for F-actin (phalloidin, red), DDR1 (green), and DNA (blue). White arrows represent areas where there are no cell–cell contacts. (B, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion of DDR1 in MCF10A cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (C, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion DDR1 in HaCaT cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (D) Western blot analysis of the levels of E-cadherin and DDR1 in RPE-1 cells expressing exogenous WT E-cadherin and E-cadherin DN. (E) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN before and after DDR1 depletion by siRNA ( n = 150). (F) Quantification of centrosome clustering in metaphase and cytokinesis in MCF10A cells treated with 15 µM DDR1 inhibitor for 3 h. (G) Western blot analysis of RhoE levels in HaCaT cells after siRNA depletion of RhoE. (H) Quantification of centrosome clustering in cytokinesis upon RhoE depletion ( n = 150). (I) Schematic representation of RhoE-mediated regulation of cortical contractility downstream of E-cadherin. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P
    Figure Legend Snippet: Cortical localization of DDR1 in cells expressing E-cadherin prevents efficient centrosome clustering. (A) Immunofluorescence images showing cortical localization of DDR1 during mitosis. Cells were stained for F-actin (phalloidin, red), DDR1 (green), and DNA (blue). White arrows represent areas where there are no cell–cell contacts. (B, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion of DDR1 in MCF10A cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (C, left) Western blot analysis of DDR1 and E-cadherin levels after siRNA depletion DDR1 in HaCaT cells. (Right) Quantification of centrosome clustering in cytokinesis upon DDR1 depletion. (D) Western blot analysis of the levels of E-cadherin and DDR1 in RPE-1 cells expressing exogenous WT E-cadherin and E-cadherin DN. (E) Quantification of centrosome clustering in cytokinesis in RPE-1 cells expressing E-cadherin and E-cadherin DN before and after DDR1 depletion by siRNA ( n = 150). (F) Quantification of centrosome clustering in metaphase and cytokinesis in MCF10A cells treated with 15 µM DDR1 inhibitor for 3 h. (G) Western blot analysis of RhoE levels in HaCaT cells after siRNA depletion of RhoE. (H) Quantification of centrosome clustering in cytokinesis upon RhoE depletion ( n = 150). (I) Schematic representation of RhoE-mediated regulation of cortical contractility downstream of E-cadherin. For all graphics, error bars represent mean ± SD from three independent experiments. ***, P

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    27) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    28) Product Images from "Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity"

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2009.03.022

    Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia
    Figure Legend Snippet: Weakened attachment of E-cadherin to cytoskeleton in 4.1R -/- gastric epithelia

    Techniques Used:

    29) Product Images from "E-cadherin represses anchorage-independent growth in sarcomas through both signaling and mechanical mechanisms"

    Article Title: E-cadherin represses anchorage-independent growth in sarcomas through both signaling and mechanical mechanisms

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0763

    E-cadherin inhibits spheroid formation by increased cell-cell adhesion. A. E-cadherin causes tighter clustering of cells during spheroid formation. B. A mechanical model illustrates the relationship between particle distance to adhesion. C. A mechanical model predicts E-cadherin drives down spheroid size through an increase in cell-cell adhesion. D-E. As predicted in the model, ectopic E-cadherin expression increases cell-cell adhesion in 143B ( D ) and U2OS ( E ) cells.
    Figure Legend Snippet: E-cadherin inhibits spheroid formation by increased cell-cell adhesion. A. E-cadherin causes tighter clustering of cells during spheroid formation. B. A mechanical model illustrates the relationship between particle distance to adhesion. C. A mechanical model predicts E-cadherin drives down spheroid size through an increase in cell-cell adhesion. D-E. As predicted in the model, ectopic E-cadherin expression increases cell-cell adhesion in 143B ( D ) and U2OS ( E ) cells.

    Techniques Used: Expressing

    Ectopic expression of E-cadherin in sarcoma cells inhibits phospho-CREB levels. A. A phospho-kinase array revealed that E-cadherin led to downregulation of phospho-CREB. B-C. E-cadherin-mediated phospho-CREB inhibition was verified by B. ELISAs and C. western blotting. D. Abrams canine osteosarcoma cells exhibited reduced phospho-CREB in E-cadherin over-expressing cells. E. QRT-PCR confirmed knockdown of CREB with two independent siRNAs. F. Western blotting to confirm knockdown of CREB in 143B cells. G. CREB knockdown led to a modest downregulation of 143B colony growth in soft agar.
    Figure Legend Snippet: Ectopic expression of E-cadherin in sarcoma cells inhibits phospho-CREB levels. A. A phospho-kinase array revealed that E-cadherin led to downregulation of phospho-CREB. B-C. E-cadherin-mediated phospho-CREB inhibition was verified by B. ELISAs and C. western blotting. D. Abrams canine osteosarcoma cells exhibited reduced phospho-CREB in E-cadherin over-expressing cells. E. QRT-PCR confirmed knockdown of CREB with two independent siRNAs. F. Western blotting to confirm knockdown of CREB in 143B cells. G. CREB knockdown led to a modest downregulation of 143B colony growth in soft agar.

    Techniques Used: Expressing, Inhibition, Western Blot, Quantitative RT-PCR

    TBX2 knockdown phenocopies E-cadherin-mediated CREB inhibition. A-B. TBX2 mRNA is downregulated in E-cadherin-expressing A. 143B cells and B. U2OS cells. C. CREB knockdown with two independent siRNAs had no effect on TBX2 mRNA. D-F. Conversely, TBX2 knockdown, verified in D. led to a significant reduction in CREB mRNA ( E ) and protein level ( F ).
    Figure Legend Snippet: TBX2 knockdown phenocopies E-cadherin-mediated CREB inhibition. A-B. TBX2 mRNA is downregulated in E-cadherin-expressing A. 143B cells and B. U2OS cells. C. CREB knockdown with two independent siRNAs had no effect on TBX2 mRNA. D-F. Conversely, TBX2 knockdown, verified in D. led to a significant reduction in CREB mRNA ( E ) and protein level ( F ).

    Techniques Used: Inhibition, Expressing

    E-cadherin inhibits anchorage-independent growth of sarcomas. A. Anchorage-independent growth of 143B cells expressing E-cadherin was significantly inhibited. B-C. E-cadherin expression leads to reduces spheroid size in B. 143B and C. U2OS cells.
    Figure Legend Snippet: E-cadherin inhibits anchorage-independent growth of sarcomas. A. Anchorage-independent growth of 143B cells expressing E-cadherin was significantly inhibited. B-C. E-cadherin expression leads to reduces spheroid size in B. 143B and C. U2OS cells.

    Techniques Used: Expressing

    Ectopic E-cadherin expression in sarcoma cells does not alter EMT. A. Ectopic expression of E-cadherin in 143B human osteosarcoma cells has no influence on mesenchymal markers (Snail, Slug, Twist, Zeb1, Vimentin). B. E-cadherin expression has no effect on migration of 143B cells. C. Images in B were collected every two hours and quantified using the IncuCyte Zoom system. D. E-cadherin expression does not change invasion in 143B cells. E. Using mRNA expression of CDH1 (E-cadherin), empirical probably density functions of EMT scores for CDH1-high (red) and CDH1-low (blue) TCGA sarcoma sample were constructed by interpolation of the EMT score histogram (E
    Figure Legend Snippet: Ectopic E-cadherin expression in sarcoma cells does not alter EMT. A. Ectopic expression of E-cadherin in 143B human osteosarcoma cells has no influence on mesenchymal markers (Snail, Slug, Twist, Zeb1, Vimentin). B. E-cadherin expression has no effect on migration of 143B cells. C. Images in B were collected every two hours and quantified using the IncuCyte Zoom system. D. E-cadherin expression does not change invasion in 143B cells. E. Using mRNA expression of CDH1 (E-cadherin), empirical probably density functions of EMT scores for CDH1-high (red) and CDH1-low (blue) TCGA sarcoma sample were constructed by interpolation of the EMT score histogram (E

    Techniques Used: Expressing, Migration, Construct

    E-cadherin upregulation is prognostic for improved outcomes in sarcoma. A-B. Osteosarcomas with elevated E-cadherin have better metastasis-free survival ( A ) and overall survival ( B ) as compared to tumors with low/no E-cadherin expression. C-D. Soft tissue sarcomas (STS) from The Cancer Genome Atlas with higher E-cadherin mRNA ( C ) and protein expression ( D ) have improved overall survival as compared to tumors with low or no E-cadherin.
    Figure Legend Snippet: E-cadherin upregulation is prognostic for improved outcomes in sarcoma. A-B. Osteosarcomas with elevated E-cadherin have better metastasis-free survival ( A ) and overall survival ( B ) as compared to tumors with low/no E-cadherin expression. C-D. Soft tissue sarcomas (STS) from The Cancer Genome Atlas with higher E-cadherin mRNA ( C ) and protein expression ( D ) have improved overall survival as compared to tumors with low or no E-cadherin.

    Techniques Used: Expressing

    Related Articles

    Clone Assay:

    Article Title: miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis
    Article Snippet: .. Constructs and transient transfections A portion of the 3’-UTR of e-cadherin was previously cloned into pmiR-Report, the seed region subsequently mutagenized [ ] and both wild type and mutated clones were obtained through Addgene (Plasmids 25038 and 25039; http://www.addgene.org ). .. Transient co-transfections of the 3’-UTR fragments cloned into the luciferase reporter vector along with a renilla luciferase vector (Promega) for normalization were performed using TransIT® -LT1 Transfection Reagent (Mirus BIO LLC) according to the manufacturer’s instructions.

    Transfection:

    Article Title: miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis
    Article Snippet: .. Constructs and transient transfections A portion of the 3’-UTR of e-cadherin was previously cloned into pmiR-Report, the seed region subsequently mutagenized [ ] and both wild type and mutated clones were obtained through Addgene (Plasmids 25038 and 25039; http://www.addgene.org ). .. Transient co-transfections of the 3’-UTR fragments cloned into the luciferase reporter vector along with a renilla luciferase vector (Promega) for normalization were performed using TransIT® -LT1 Transfection Reagent (Mirus BIO LLC) according to the manufacturer’s instructions.

    Amplification:

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: .. The templates used for the amplification of β-catenin, α-catenin and E-cadherin were from Addgene, GeneCopoeia or Origene respectively. ..

    Construct:

    Article Title: miR-9 Acts as an OncomiR in Prostate Cancer through Multiple Pathways That Drive Tumour Progression and Metastasis
    Article Snippet: .. Constructs and transient transfections A portion of the 3’-UTR of e-cadherin was previously cloned into pmiR-Report, the seed region subsequently mutagenized [ ] and both wild type and mutated clones were obtained through Addgene (Plasmids 25038 and 25039; http://www.addgene.org ). .. Transient co-transfections of the 3’-UTR fragments cloned into the luciferase reporter vector along with a renilla luciferase vector (Promega) for normalization were performed using TransIT® -LT1 Transfection Reagent (Mirus BIO LLC) according to the manufacturer’s instructions.

    Purification:

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: .. The His-tagged 4.1R and its domains were purified by Nickle column and the GST-tagged β-catenin, α-catenin and cytoplasmic domain of E-cadherin were purified by glutathione sepharose-4B affinity column. .. To examine the binding of 4.1R or its domains to β-catenin, α-catenin or E-cadherin, GST, GST-tagged β-catenin, α-catenin or cytoplasmic domain of E-cadherin was coupled to glutathione sepharose-4B beads at room temperature for 30 min. Beads were pelleted and washed.

    Generated:

    Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer
    Article Snippet: .. Split luciferase assay Fusion cDNAs were generated by deleting the stop codon in human Cx43 (Origene), PCDH7 (Origene), E-cadherin (Addgene) or N-cadherin (Addgene) cDNAs and splicing the N-terminal or C-terminal fragment of firefly luciferase . (Addgene). .. Constructs were cloned into pLVX lentiviral expression vector and transduced into non-GFP-luciferase-labeled parental MDA-MB-231 or H2030 cells.

    Luciferase:

    Article Title: Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer
    Article Snippet: .. Split luciferase assay Fusion cDNAs were generated by deleting the stop codon in human Cx43 (Origene), PCDH7 (Origene), E-cadherin (Addgene) or N-cadherin (Addgene) cDNAs and splicing the N-terminal or C-terminal fragment of firefly luciferase . (Addgene). .. Constructs were cloned into pLVX lentiviral expression vector and transduced into non-GFP-luciferase-labeled parental MDA-MB-231 or H2030 cells.

    Affinity Column:

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: .. The His-tagged 4.1R and its domains were purified by Nickle column and the GST-tagged β-catenin, α-catenin and cytoplasmic domain of E-cadherin were purified by glutathione sepharose-4B affinity column. .. To examine the binding of 4.1R or its domains to β-catenin, α-catenin or E-cadherin, GST, GST-tagged β-catenin, α-catenin or cytoplasmic domain of E-cadherin was coupled to glutathione sepharose-4B beads at room temperature for 30 min. Beads were pelleted and washed.

    other:

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: The current view about the connection between E-cadherin/catenin complexes and the cytoskeleton is that E-cadherin binds to β-catenin which in turn is linked to the actin skeleton through its interaction with α-catenin.

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion.

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: As shown in , 4.1R bound to the GST-tagged β-catenin, but not to GST-tagged α-catenin, cytoplasmic domain of E-cadherin or GST alone.

    Article Title: Protein 4.1R links E-cadherin/β-catenin complex to the cytoskeleton through its direct interaction with β-catenin and modulates adherens junction integrity
    Article Snippet: β-catenin plays an important role in linking E-cadherin to the actin cytoskeleton [ - ].

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    Addgene inc gfp fused ve cadherin
    Melanoma cells underwent TEM in plasma via paracellular routes. A, confocal images show the migration of melanoma cells from the apical side to the basolateral side of HUVECs in the presence or absence of PFP. The locations of the same Lu1205 cells were captured at 0, 15, 30, 45, and 60 min of TEM. Lu1205 cells were stained with DiI before being seeded on a HUVEC monolayer, which was transfected with <t>GFP-VE-cadherin.</t> B, X/Z cross-sections of images of transmigrating Lu1205 cells. Micrographs show the stages of TEM as follows: TC settlement on EC; TC margination through EC; and TC localization beneath EC. The schematic drawing above each image represents the stage of TEM shown in the micrograph. Bar, 10 μm. White arrowheads indicate junctional sites. C, percentage of adherent Lu1205 cells taking transcellular and paracellular routes for TEM in the presence or absence of PFP. D, percentage of buffer, scrambled siRNA, or siB-Raf(V600E)-transfected Lu1205 cells taking transcellular and paracellular routes for TEM in the presence of PFP. E, percentage of Lu1205 cells taking transcellular and paracellular routes for migrating across buffer, scrambled siRNA, or siPAR-1-transfected HUVEC monolayer in the presence of PFP. Values are mean ± S.E. from three independent experiments. **, p
    Gfp Fused Ve Cadherin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Establishing a functional link between <t>E-cadherin</t> and α-catenin with optochemical dimerizers. a , b Immunofluorescence images of A431 α-catenin KO cells coexpressing <t>E-cadherin-Δcyto-GFP-Halo</t> and SNAP-mCherry-ΔN-α-catenin. Without dimerizer E-cadherin shows diffuse membrane localization, whereas α-catenin and β-catenin are cytosolic. Dimerizer induced E-cadherin-α-catenin complex formation indirectly recruits also β-catenin and causes rearrangements of actin fibers. 365 nm light disseminates Ha-pl-BG treated cells but not Ha-BG treated cells. Scale bars 20 µm. c Western Blot analysis of E-cadherin-α-catenin complexes. Without dimerizer (lane 1) only the 119 kDa SNAP-(mCherry)-α-catenin is detected, whereas with dimerizer (lane 2 and 4) an additional band of 265 kDa is detected, resembling the hetero-dimer E-cadherin-α-catenin complex. After 365 nm light, the heavy band disappears in Ha-pl-BG treated cells but not in Ha-BG samples (lane 3 and 5). β-actin serves as loading control. d Application of LInDA to dissociate AJs in MDA-MB-468 epithelial cancer cells coexpressing E-cadherin-Δcyto-GFP-Halo and SNAP-mCherry-ΔN-α-catenin. Filamentous actin was labeled using phalloidin conjugated with Alexa647 dye. In absence of the dimerizer, punctate E-cadherin clusters are present at the cell membrane, whereas α-catenin and β-catenin are diffuse in the cytoplasm. Addition of Ha-pl-BG induces E-cadherin mediated AJ formation by recruiting the cytosolic α-catenin and β-catenin to the cell membrane. Scale bar 20 µm.
    E Cadherin Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    <t>E-cadherin</t> is required for force transmission between CAFs and A431 cells (a) Net force transmitted between CAFs and A431 cells before the onset of contact and during contact. Experiments were performed under control conditions and after depletion of E-cadherin in the A431 cells using CRISPR/Cas9. The white bar indicates background noise levels. (-/+) n=12 CAFs from 9 independent experiments, (+/+) n=13 CAFs from 9 independent experiments, (-/-) n=13 CAFs from 2 independent experiments, (+/-) n=17 CAFs from 2 independent experiments, n=13 image regions from 8 independent experiments (noise level). Error bars represent s.e.m. *** indicates p
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    Melanoma cells underwent TEM in plasma via paracellular routes. A, confocal images show the migration of melanoma cells from the apical side to the basolateral side of HUVECs in the presence or absence of PFP. The locations of the same Lu1205 cells were captured at 0, 15, 30, 45, and 60 min of TEM. Lu1205 cells were stained with DiI before being seeded on a HUVEC monolayer, which was transfected with GFP-VE-cadherin. B, X/Z cross-sections of images of transmigrating Lu1205 cells. Micrographs show the stages of TEM as follows: TC settlement on EC; TC margination through EC; and TC localization beneath EC. The schematic drawing above each image represents the stage of TEM shown in the micrograph. Bar, 10 μm. White arrowheads indicate junctional sites. C, percentage of adherent Lu1205 cells taking transcellular and paracellular routes for TEM in the presence or absence of PFP. D, percentage of buffer, scrambled siRNA, or siB-Raf(V600E)-transfected Lu1205 cells taking transcellular and paracellular routes for TEM in the presence of PFP. E, percentage of Lu1205 cells taking transcellular and paracellular routes for migrating across buffer, scrambled siRNA, or siPAR-1-transfected HUVEC monolayer in the presence of PFP. Values are mean ± S.E. from three independent experiments. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Mutant B-Raf(V600E) Promotes Melanoma Paracellular Transmigration by Inducing Thrombin-mediated Endothelial Junction Breakdown *

    doi: 10.1074/jbc.M115.696419

    Figure Lengend Snippet: Melanoma cells underwent TEM in plasma via paracellular routes. A, confocal images show the migration of melanoma cells from the apical side to the basolateral side of HUVECs in the presence or absence of PFP. The locations of the same Lu1205 cells were captured at 0, 15, 30, 45, and 60 min of TEM. Lu1205 cells were stained with DiI before being seeded on a HUVEC monolayer, which was transfected with GFP-VE-cadherin. B, X/Z cross-sections of images of transmigrating Lu1205 cells. Micrographs show the stages of TEM as follows: TC settlement on EC; TC margination through EC; and TC localization beneath EC. The schematic drawing above each image represents the stage of TEM shown in the micrograph. Bar, 10 μm. White arrowheads indicate junctional sites. C, percentage of adherent Lu1205 cells taking transcellular and paracellular routes for TEM in the presence or absence of PFP. D, percentage of buffer, scrambled siRNA, or siB-Raf(V600E)-transfected Lu1205 cells taking transcellular and paracellular routes for TEM in the presence of PFP. E, percentage of Lu1205 cells taking transcellular and paracellular routes for migrating across buffer, scrambled siRNA, or siPAR-1-transfected HUVEC monolayer in the presence of PFP. Values are mean ± S.E. from three independent experiments. **, p

    Article Snippet: GFP-fused VE-cadherin was generated by cloning mApple-VE-cadherin (Addgene) to GFP-pcDNA3 with BamHI and XbaI restriction sites.

    Techniques: Transmission Electron Microscopy, Migration, Staining, Transfection

    Establishing a functional link between E-cadherin and α-catenin with optochemical dimerizers. a , b Immunofluorescence images of A431 α-catenin KO cells coexpressing E-cadherin-Δcyto-GFP-Halo and SNAP-mCherry-ΔN-α-catenin. Without dimerizer E-cadherin shows diffuse membrane localization, whereas α-catenin and β-catenin are cytosolic. Dimerizer induced E-cadherin-α-catenin complex formation indirectly recruits also β-catenin and causes rearrangements of actin fibers. 365 nm light disseminates Ha-pl-BG treated cells but not Ha-BG treated cells. Scale bars 20 µm. c Western Blot analysis of E-cadherin-α-catenin complexes. Without dimerizer (lane 1) only the 119 kDa SNAP-(mCherry)-α-catenin is detected, whereas with dimerizer (lane 2 and 4) an additional band of 265 kDa is detected, resembling the hetero-dimer E-cadherin-α-catenin complex. After 365 nm light, the heavy band disappears in Ha-pl-BG treated cells but not in Ha-BG samples (lane 3 and 5). β-actin serves as loading control. d Application of LInDA to dissociate AJs in MDA-MB-468 epithelial cancer cells coexpressing E-cadherin-Δcyto-GFP-Halo and SNAP-mCherry-ΔN-α-catenin. Filamentous actin was labeled using phalloidin conjugated with Alexa647 dye. In absence of the dimerizer, punctate E-cadherin clusters are present at the cell membrane, whereas α-catenin and β-catenin are diffuse in the cytoplasm. Addition of Ha-pl-BG induces E-cadherin mediated AJ formation by recruiting the cytosolic α-catenin and β-catenin to the cell membrane. Scale bar 20 µm.

    Journal: Nature Communications

    Article Title: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells

    doi: 10.1038/s41467-020-14390-1

    Figure Lengend Snippet: Establishing a functional link between E-cadherin and α-catenin with optochemical dimerizers. a , b Immunofluorescence images of A431 α-catenin KO cells coexpressing E-cadherin-Δcyto-GFP-Halo and SNAP-mCherry-ΔN-α-catenin. Without dimerizer E-cadherin shows diffuse membrane localization, whereas α-catenin and β-catenin are cytosolic. Dimerizer induced E-cadherin-α-catenin complex formation indirectly recruits also β-catenin and causes rearrangements of actin fibers. 365 nm light disseminates Ha-pl-BG treated cells but not Ha-BG treated cells. Scale bars 20 µm. c Western Blot analysis of E-cadherin-α-catenin complexes. Without dimerizer (lane 1) only the 119 kDa SNAP-(mCherry)-α-catenin is detected, whereas with dimerizer (lane 2 and 4) an additional band of 265 kDa is detected, resembling the hetero-dimer E-cadherin-α-catenin complex. After 365 nm light, the heavy band disappears in Ha-pl-BG treated cells but not in Ha-BG samples (lane 3 and 5). β-actin serves as loading control. d Application of LInDA to dissociate AJs in MDA-MB-468 epithelial cancer cells coexpressing E-cadherin-Δcyto-GFP-Halo and SNAP-mCherry-ΔN-α-catenin. Filamentous actin was labeled using phalloidin conjugated with Alexa647 dye. In absence of the dimerizer, punctate E-cadherin clusters are present at the cell membrane, whereas α-catenin and β-catenin are diffuse in the cytoplasm. Addition of Ha-pl-BG induces E-cadherin mediated AJ formation by recruiting the cytosolic α-catenin and β-catenin to the cell membrane. Scale bar 20 µm.

    Article Snippet: E-cadherin-GFP was a gift from Jennifer Stow (Addgene plasmid # 28009; http://n2t.net/addgene:28009 ; RRID:Addgene_28009) .

    Techniques: Functional Assay, Immunofluorescence, Western Blot, Multiple Displacement Amplification, Labeling

    Dimerizer-mediated reconstitution followed by LInDA to specifically target AJs. a When coexpressed in α-catenin KO cells, the α-catenin (mCherry-labled, shown in red) construct is cytosolic, whereas E-cadherin (GFP-labled, shown in green) is located at the cell membrane forming unstable complexes at cell–cell-interfaces. Following addition of Ha-pl-BG (−9:00 h) AJs form (−4:00 h) and mature into defined linear structures accompanied by cell compaction (before). After a short pulse of 350 nm light the E-cadherin-α-catenin complex disassembles and α-catenin becomes cytosolic again (after and 4:00 h). E-cadherin clusters destabilize and cells disseminate. Scale bar 20 µm. b Normalized profiles for GFP (green) and mCherry (red) fluorescence intensity in cross-sections of cell–cell contacts as measures for localization of E-cadherin and α-catenin, respectively. A representative cross-section is shown by the arrow headed dashed lines in a . The lines have been repositioned for each time point to reflect profiles perpendicular and centered to the cell–cell interface. Mean ± s.d. are shown for n = 45 cross-sections in multiple fields of view for each time point. c Staining for desmosome proteins reveals the presence of desmoplakin at cell–cell contacts only in presence of the dimerizer and in cell tethers following the light-induced dissociation of AJs. Scale bar 20 µm for single color images and overlay image, 5 µm for 5× zoom image.

    Journal: Nature Communications

    Article Title: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells

    doi: 10.1038/s41467-020-14390-1

    Figure Lengend Snippet: Dimerizer-mediated reconstitution followed by LInDA to specifically target AJs. a When coexpressed in α-catenin KO cells, the α-catenin (mCherry-labled, shown in red) construct is cytosolic, whereas E-cadherin (GFP-labled, shown in green) is located at the cell membrane forming unstable complexes at cell–cell-interfaces. Following addition of Ha-pl-BG (−9:00 h) AJs form (−4:00 h) and mature into defined linear structures accompanied by cell compaction (before). After a short pulse of 350 nm light the E-cadherin-α-catenin complex disassembles and α-catenin becomes cytosolic again (after and 4:00 h). E-cadherin clusters destabilize and cells disseminate. Scale bar 20 µm. b Normalized profiles for GFP (green) and mCherry (red) fluorescence intensity in cross-sections of cell–cell contacts as measures for localization of E-cadherin and α-catenin, respectively. A representative cross-section is shown by the arrow headed dashed lines in a . The lines have been repositioned for each time point to reflect profiles perpendicular and centered to the cell–cell interface. Mean ± s.d. are shown for n = 45 cross-sections in multiple fields of view for each time point. c Staining for desmosome proteins reveals the presence of desmoplakin at cell–cell contacts only in presence of the dimerizer and in cell tethers following the light-induced dissociation of AJs. Scale bar 20 µm for single color images and overlay image, 5 µm for 5× zoom image.

    Article Snippet: E-cadherin-GFP was a gift from Jennifer Stow (Addgene plasmid # 28009; http://n2t.net/addgene:28009 ; RRID:Addgene_28009) .

    Techniques: Construct, Fluorescence, Staining

    Multiscale targeting of AJs assembly and disassembly with high spatial precision. a For visualization in live cell experiments, E-cadherin (shown in green) and α-catenin (shown in red) are tagged with GFP and mCherry, respectively. Targeted dissociation of Ha-pl-BG reconstituted AJs with subcellular precision before (upper row) directly after (middle) and 5 min after 405 nm laser area scanning (white dashed rectangular in the overlay). Scale bar 10 µm. b Kymograph analysis of E-cadherin and α-catenin intensities of targeted (line 1) and untargeted (line 2) AJs in a . The time point of laser scanning is indicated as black and white dashed lines. Scale as indicated by arrows in the lower right. c Monolayer compaction via AJ reconstitution after addition of Ha-pl-BG leads to reintegration of the loosely attached cells in a monolayer. After 4 h, the white dashed outlined area was scanned with a 405 nm laser. Note that only cells in the targeted area change their morphology and push out excess cells immediately. Scale bar 200 µm.

    Journal: Nature Communications

    Article Title: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells

    doi: 10.1038/s41467-020-14390-1

    Figure Lengend Snippet: Multiscale targeting of AJs assembly and disassembly with high spatial precision. a For visualization in live cell experiments, E-cadherin (shown in green) and α-catenin (shown in red) are tagged with GFP and mCherry, respectively. Targeted dissociation of Ha-pl-BG reconstituted AJs with subcellular precision before (upper row) directly after (middle) and 5 min after 405 nm laser area scanning (white dashed rectangular in the overlay). Scale bar 10 µm. b Kymograph analysis of E-cadherin and α-catenin intensities of targeted (line 1) and untargeted (line 2) AJs in a . The time point of laser scanning is indicated as black and white dashed lines. Scale as indicated by arrows in the lower right. c Monolayer compaction via AJ reconstitution after addition of Ha-pl-BG leads to reintegration of the loosely attached cells in a monolayer. After 4 h, the white dashed outlined area was scanned with a 405 nm laser. Note that only cells in the targeted area change their morphology and push out excess cells immediately. Scale bar 200 µm.

    Article Snippet: E-cadherin-GFP was a gift from Jennifer Stow (Addgene plasmid # 28009; http://n2t.net/addgene:28009 ; RRID:Addgene_28009) .

    Techniques:

    ExlA-dependent cleavage of E- and VE-cadherins. A . A549 cells (left) or HUVECs (right) were incubated with various P . aeruginosa strains: CHA, PAO1F or CLJ1, or were mock-infected with LB (NI). Cell extracts were prepared at different times post-infection, as indicated and analysed by Western blot using E-cadherin (left) or VE-cadherin (right) antibodies. In both cases, the full-length (FL) and post-cleavage C-terminal (CTF) fragments are shown. β-actin was used as loading control. The experiment was performed 3 times for A549 and twice for HUVECs with similar results. B . A549 cells (left) and HUVECs (right) were incubated with IHMA87, IHMA87Δ exlA or IHMA87Δ exlA/exlA strains, and cellular extracts were analysed as above. The experiment was performed 3 times for A549 and once for HUVECs. C . Similar experiments with A549 cells (left) or HUVECs (right) incubated with E . coli containing the empty vector ( exlBA -) or E-coli expressing ExlB-ExlA (exlBA +) . D . A549-E-cadherin-GFP cells were incubated with CLJ1, IHMA87, IHMA87Δ exlA or PAO1F bacteria. E-cadherin-GFP (green) as well as nuclei labelling by propidium iodide (red) were followed by confocal videomicroscopy. Times post-infection are shown as “h:min”. One z-position is represented. The experiment was performed twice, with 4–5 positions recorded each time. All films showed similar results. E . Mice (5 per condition) were infected by bacteria inhalation (2.5x10 6 ), using CLJ1 strain, or were mock-infected with PBS (NI). Mice were euthanized at 18 h.p.i.; protein extracts were prepared from lungs and were analysed by Western blot using E- and VE-cadherin antibodies (left). Histograms (right) show the E-cadherin/ β-actin and VE-cadherin/ β-actin ratios of band intensities, represented as means + s.d. Significance was calculated using Mann-Whitney’s test, as variances were not equal. The experiment was repeated once, with similar results.

    Journal: PLoS Pathogens

    Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

    doi: 10.1371/journal.ppat.1006579

    Figure Lengend Snippet: ExlA-dependent cleavage of E- and VE-cadherins. A . A549 cells (left) or HUVECs (right) were incubated with various P . aeruginosa strains: CHA, PAO1F or CLJ1, or were mock-infected with LB (NI). Cell extracts were prepared at different times post-infection, as indicated and analysed by Western blot using E-cadherin (left) or VE-cadherin (right) antibodies. In both cases, the full-length (FL) and post-cleavage C-terminal (CTF) fragments are shown. β-actin was used as loading control. The experiment was performed 3 times for A549 and twice for HUVECs with similar results. B . A549 cells (left) and HUVECs (right) were incubated with IHMA87, IHMA87Δ exlA or IHMA87Δ exlA/exlA strains, and cellular extracts were analysed as above. The experiment was performed 3 times for A549 and once for HUVECs. C . Similar experiments with A549 cells (left) or HUVECs (right) incubated with E . coli containing the empty vector ( exlBA -) or E-coli expressing ExlB-ExlA (exlBA +) . D . A549-E-cadherin-GFP cells were incubated with CLJ1, IHMA87, IHMA87Δ exlA or PAO1F bacteria. E-cadherin-GFP (green) as well as nuclei labelling by propidium iodide (red) were followed by confocal videomicroscopy. Times post-infection are shown as “h:min”. One z-position is represented. The experiment was performed twice, with 4–5 positions recorded each time. All films showed similar results. E . Mice (5 per condition) were infected by bacteria inhalation (2.5x10 6 ), using CLJ1 strain, or were mock-infected with PBS (NI). Mice were euthanized at 18 h.p.i.; protein extracts were prepared from lungs and were analysed by Western blot using E- and VE-cadherin antibodies (left). Histograms (right) show the E-cadherin/ β-actin and VE-cadherin/ β-actin ratios of band intensities, represented as means + s.d. Significance was calculated using Mann-Whitney’s test, as variances were not equal. The experiment was repeated once, with similar results.

    Article Snippet: A549 E-cadherin GFP cells were obtained after transfection with pCDNA3.1-E-cadherin-GFP (Addgene) using Lipofectamine 2000 (Life Technologies).

    Techniques: Incubation, Infection, Western Blot, Plasmid Preparation, Expressing, Mouse Assay, MANN-WHITNEY

    Role of calcium influx in cadherin cleavage and necrosis. A . A549 cells were incubated with various concentrations of TFP, as indicated, to impede calmodulin interaction with ADAM10. Ionomycin was used as positive controls. E-cadherin cleavage was assessed by Western blot. The experiment was performed twice. B . Western blot analysis of A549 E-cadherin contents after infection with CLJ1 or IHMA87, in presence or absence of BAPTA-AM. Both experiments were performed 3 times. C . LDH release of A549 cells infected with either CLJ1 or IHMA87, in presence/ absence of BAPTA-AM. Student’s t-test showed significance between the two treatments for both CLJ1 and IHMA87 data (p-values indicated above the bars). The experiment was performed 3 times.

    Journal: PLoS Pathogens

    Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

    doi: 10.1371/journal.ppat.1006579

    Figure Lengend Snippet: Role of calcium influx in cadherin cleavage and necrosis. A . A549 cells were incubated with various concentrations of TFP, as indicated, to impede calmodulin interaction with ADAM10. Ionomycin was used as positive controls. E-cadherin cleavage was assessed by Western blot. The experiment was performed twice. B . Western blot analysis of A549 E-cadherin contents after infection with CLJ1 or IHMA87, in presence or absence of BAPTA-AM. Both experiments were performed 3 times. C . LDH release of A549 cells infected with either CLJ1 or IHMA87, in presence/ absence of BAPTA-AM. Student’s t-test showed significance between the two treatments for both CLJ1 and IHMA87 data (p-values indicated above the bars). The experiment was performed 3 times.

    Article Snippet: A549 E-cadherin GFP cells were obtained after transfection with pCDNA3.1-E-cadherin-GFP (Addgene) using Lipofectamine 2000 (Life Technologies).

    Techniques: Incubation, Western Blot, Infection

    E-cadherin is required for force transmission between CAFs and A431 cells (a) Net force transmitted between CAFs and A431 cells before the onset of contact and during contact. Experiments were performed under control conditions and after depletion of E-cadherin in the A431 cells using CRISPR/Cas9. The white bar indicates background noise levels. (-/+) n=12 CAFs from 9 independent experiments, (+/+) n=13 CAFs from 9 independent experiments, (-/-) n=13 CAFs from 2 independent experiments, (+/-) n=17 CAFs from 2 independent experiments, n=13 image regions from 8 independent experiments (noise level). Error bars represent s.e.m. *** indicates p

    Journal: Nature cell biology

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    doi: 10.1038/ncb3478

    Figure Lengend Snippet: E-cadherin is required for force transmission between CAFs and A431 cells (a) Net force transmitted between CAFs and A431 cells before the onset of contact and during contact. Experiments were performed under control conditions and after depletion of E-cadherin in the A431 cells using CRISPR/Cas9. The white bar indicates background noise levels. (-/+) n=12 CAFs from 9 independent experiments, (+/+) n=13 CAFs from 9 independent experiments, (-/-) n=13 CAFs from 2 independent experiments, (+/-) n=17 CAFs from 2 independent experiments, n=13 image regions from 8 independent experiments (noise level). Error bars represent s.e.m. *** indicates p

    Article Snippet: To visualize N-cadherin in CAFs and E-cadherin in A431 cells during time lapse experiments, unless stated otherwise cells were transfected with the N-cadherin-EGFP Plasmid (Addgene, #18870) and E-cadherin Ruby Plasmid (from Kurt Anderson’s lab) respectively two days before experiments using the Neon transfection device according to the manufacturer’s instructions (Invitrogen).

    Techniques: Transmission Assay, CRISPR

    The E-cadherin/N-cadherin junction enables collective cancer cell invasion in 3D (a-e) Fluorescence images of spheroids containing different mixtures of CAFs and A431 cells after 60 hours of invasion in an organotypic ECM. (a) 1:1 mixture of control A431 (YPet) and control CAFs (KEIMA). (b) 1:1 mixture of A431-EcadKO (mCherry) and control CAFs (KEIMA). (c) 1:1:2 mixture of A431 control (YPet), A431-EcadKO (mCherry), and control CAFs (KEIMA). Arrow points to one A431-EcadKO cell in the invasive strand. (d) 1:1 mixture of A431 control (YPet) and CAFs-siRNA (KEIMA). (e) for additional spheroid conditions. Scale bars, 100 µm. (f) Average number of strands per spheroid in the conditions shown in (a-e) , and CAF-siCT and A431-αcatWT. Number of spheroids measured: n=24 (control), n=18 (EKO, P

    Journal: Nature cell biology

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    doi: 10.1038/ncb3478

    Figure Lengend Snippet: The E-cadherin/N-cadherin junction enables collective cancer cell invasion in 3D (a-e) Fluorescence images of spheroids containing different mixtures of CAFs and A431 cells after 60 hours of invasion in an organotypic ECM. (a) 1:1 mixture of control A431 (YPet) and control CAFs (KEIMA). (b) 1:1 mixture of A431-EcadKO (mCherry) and control CAFs (KEIMA). (c) 1:1:2 mixture of A431 control (YPet), A431-EcadKO (mCherry), and control CAFs (KEIMA). Arrow points to one A431-EcadKO cell in the invasive strand. (d) 1:1 mixture of A431 control (YPet) and CAFs-siRNA (KEIMA). (e) for additional spheroid conditions. Scale bars, 100 µm. (f) Average number of strands per spheroid in the conditions shown in (a-e) , and CAF-siCT and A431-αcatWT. Number of spheroids measured: n=24 (control), n=18 (EKO, P

    Article Snippet: To visualize N-cadherin in CAFs and E-cadherin in A431 cells during time lapse experiments, unless stated otherwise cells were transfected with the N-cadherin-EGFP Plasmid (Addgene, #18870) and E-cadherin Ruby Plasmid (from Kurt Anderson’s lab) respectively two days before experiments using the Neon transfection device according to the manufacturer’s instructions (Invitrogen).

    Techniques: Fluorescence

    Afadin and nectins 2 and 3 are required for CAF-led migration of cancer cells and for CAF polarization. (a) Confocal images of nectin-3 (blue), N-cadherin (green), E-cadherin (red) in a co-culture of CAFs and A431 cells (upper panels); nectin-2 (blue), N-cadherin (green), E-cadherin (red) (middle panels); afadin (blue), N-cadherin (green), E-cadherin (red) (lower panels). Yellow arrows show the localization of the CAF/A431 cell contact. Images representative of 2 samples. Scale bars, 5 µm. (b) Staining of afadin (green), and p120catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell. Images representative of 5 samples. Scale bar is 10μm. (c) Fraction of “leaders” vs “loners” in CAF-siCT (n=90 CAFs) and CAF-siAF (n=95 CAFs). Data pooled from 3 independent experiments. *** indicates P

    Journal: Nature cell biology

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    doi: 10.1038/ncb3478

    Figure Lengend Snippet: Afadin and nectins 2 and 3 are required for CAF-led migration of cancer cells and for CAF polarization. (a) Confocal images of nectin-3 (blue), N-cadherin (green), E-cadherin (red) in a co-culture of CAFs and A431 cells (upper panels); nectin-2 (blue), N-cadherin (green), E-cadherin (red) (middle panels); afadin (blue), N-cadherin (green), E-cadherin (red) (lower panels). Yellow arrows show the localization of the CAF/A431 cell contact. Images representative of 2 samples. Scale bars, 5 µm. (b) Staining of afadin (green), and p120catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell. Images representative of 5 samples. Scale bar is 10μm. (c) Fraction of “leaders” vs “loners” in CAF-siCT (n=90 CAFs) and CAF-siAF (n=95 CAFs). Data pooled from 3 independent experiments. *** indicates P

    Article Snippet: To visualize N-cadherin in CAFs and E-cadherin in A431 cells during time lapse experiments, unless stated otherwise cells were transfected with the N-cadherin-EGFP Plasmid (Addgene, #18870) and E-cadherin Ruby Plasmid (from Kurt Anderson’s lab) respectively two days before experiments using the Neon transfection device according to the manufacturer’s instructions (Invitrogen).

    Techniques: Migration, Co-Culture Assay, Staining

    CAFs and A431 cells form heterophilic E-cadherin/N-cadherin junctions (a) TEM image of contact (white arrows) between a CAF and a A431 cell. Image representative of 20 contacts from 3 independent experiments. Scale bar 100nm. (b) mRNA expression levels of E-,N- and P-cadherin in CAFs and A431 cells measured using QRT-PCR. Bars show average of technical triplicates. (c) Confocal immunofluorescence images of N-cadherin (red), E-cadherin (green), and CAGAP-mcherry (constitutively expressed by CAFs as a marker) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (d) Confocal immunofluorescence images of N-cadherin, P-cadherin, and CAGAP-mcherry (CAFs) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (e) SIM immunofluorescence images of N-cadherin (green), E-cadherin (yellow), β-catenin (red) and F-actin (blue) at a contact between CAF and A431 cell. Image representative of 15 samples. Scale bar is 1μm for zoomed areas, 10μm for merged overview projection . (f) STORM image of N-cadherin/E-cadherin localization at the contact between CAF and A431 cell. Image representative of 3 samples. Scale bar, 500nm. (g) Time-lapse images of a CAF expressing N-cadherin-GFP contacting A431 cells expressing E-cadherin-WT (red) (upper panels) or A431 cells expressing E-cadherin-W2A mutant (red) (lower panels), scale bars, 20µm . (h) Stacked histogram of life-time of the E-cadherin/N-cadherin junction (based on the E-Cadherin and N-cadherin fluorescent signals) at the contact between CAFs and A431 cells, for CAFs mixed with A431-EcadWT cells (rescue control, n=14 contacts from 3 independent experiments) and A431-EcadW2A mutant cells (n=28 contacts from 3 independent experiments). Data are pooled in three categories of contact life-time, from 0 to 30 min, from 30 to 60 min, and longer than 60 min duration. *** indicates p=0.0007, Chi-squared test.

    Journal: Nature cell biology

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    doi: 10.1038/ncb3478

    Figure Lengend Snippet: CAFs and A431 cells form heterophilic E-cadherin/N-cadherin junctions (a) TEM image of contact (white arrows) between a CAF and a A431 cell. Image representative of 20 contacts from 3 independent experiments. Scale bar 100nm. (b) mRNA expression levels of E-,N- and P-cadherin in CAFs and A431 cells measured using QRT-PCR. Bars show average of technical triplicates. (c) Confocal immunofluorescence images of N-cadherin (red), E-cadherin (green), and CAGAP-mcherry (constitutively expressed by CAFs as a marker) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (d) Confocal immunofluorescence images of N-cadherin, P-cadherin, and CAGAP-mcherry (CAFs) in a co-culture of CAFs and A431 cells. Image representative of > 4 samples. Scale bar, 5 µm. (e) SIM immunofluorescence images of N-cadherin (green), E-cadherin (yellow), β-catenin (red) and F-actin (blue) at a contact between CAF and A431 cell. Image representative of 15 samples. Scale bar is 1μm for zoomed areas, 10μm for merged overview projection . (f) STORM image of N-cadherin/E-cadherin localization at the contact between CAF and A431 cell. Image representative of 3 samples. Scale bar, 500nm. (g) Time-lapse images of a CAF expressing N-cadherin-GFP contacting A431 cells expressing E-cadherin-WT (red) (upper panels) or A431 cells expressing E-cadherin-W2A mutant (red) (lower panels), scale bars, 20µm . (h) Stacked histogram of life-time of the E-cadherin/N-cadherin junction (based on the E-Cadherin and N-cadherin fluorescent signals) at the contact between CAFs and A431 cells, for CAFs mixed with A431-EcadWT cells (rescue control, n=14 contacts from 3 independent experiments) and A431-EcadW2A mutant cells (n=28 contacts from 3 independent experiments). Data are pooled in three categories of contact life-time, from 0 to 30 min, from 30 to 60 min, and longer than 60 min duration. *** indicates p=0.0007, Chi-squared test.

    Article Snippet: To visualize N-cadherin in CAFs and E-cadherin in A431 cells during time lapse experiments, unless stated otherwise cells were transfected with the N-cadherin-EGFP Plasmid (Addgene, #18870) and E-cadherin Ruby Plasmid (from Kurt Anderson’s lab) respectively two days before experiments using the Neon transfection device according to the manufacturer’s instructions (Invitrogen).

    Techniques: Transmission Electron Microscopy, Expressing, Quantitative RT-PCR, Immunofluorescence, Marker, Co-Culture Assay, Mutagenesis

    Evidence of E-cadherin/N-cadherin junctions in lung adenocarcinoma and vulval squamous cell carcinoma (a,b) for a third patient). (c) Immunostaining of the contact between cancer cells and CAFs both isolated from one patient with vulval squamous cell carcinoma. N-cadherin (red), E-cadherin (green), F-actin (blue). Images representative of 2 patient samples. Scale bar, 5µm. (d) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431 (green), CAF (red), and collagen second harmonic (magenta), arrows highlight the different tumor components. Images representative of 3 samples. Scale bar is 20μm. (e) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431-Ecad-Ruby and vulval CAF-Ncad-GFP. White arrow highlights heterotypic contact. Images representative of 3 samples. Scale bar is 20μm. (f) Images show staining of F-actin (blue), E-cadherin (green), and αSMA (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, v - vessel. Images representative of 5 samples. Scale bar, 10μm. (g) Staining of fibronectin (magenta), active integrin β1 (green), and β-catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, yellow arrow highlights integrin/ECM contact by CAF, BM – basement membrane. Images representative of 5 samples. Scale bar, 10μm.

    Journal: Nature cell biology

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    doi: 10.1038/ncb3478

    Figure Lengend Snippet: Evidence of E-cadherin/N-cadherin junctions in lung adenocarcinoma and vulval squamous cell carcinoma (a,b) for a third patient). (c) Immunostaining of the contact between cancer cells and CAFs both isolated from one patient with vulval squamous cell carcinoma. N-cadherin (red), E-cadherin (green), F-actin (blue). Images representative of 2 patient samples. Scale bar, 5µm. (d) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431 (green), CAF (red), and collagen second harmonic (magenta), arrows highlight the different tumor components. Images representative of 3 samples. Scale bar is 20μm. (e) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431-Ecad-Ruby and vulval CAF-Ncad-GFP. White arrow highlights heterotypic contact. Images representative of 3 samples. Scale bar is 20μm. (f) Images show staining of F-actin (blue), E-cadherin (green), and αSMA (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, v - vessel. Images representative of 5 samples. Scale bar, 10μm. (g) Staining of fibronectin (magenta), active integrin β1 (green), and β-catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, yellow arrow highlights integrin/ECM contact by CAF, BM – basement membrane. Images representative of 5 samples. Scale bar, 10μm.

    Article Snippet: To visualize N-cadherin in CAFs and E-cadherin in A431 cells during time lapse experiments, unless stated otherwise cells were transfected with the N-cadherin-EGFP Plasmid (Addgene, #18870) and E-cadherin Ruby Plasmid (from Kurt Anderson’s lab) respectively two days before experiments using the Neon transfection device according to the manufacturer’s instructions (Invitrogen).

    Techniques: Immunostaining, Isolation, Imaging, Staining

    Heterophilic E-cadherin/N-cadherin junctions withstand forces and trigger mechanotransduction (a) Illustration of the magnetic tweezers experimental setup. (b) Bead detachment data in A431 cells (CT), A431-EcadKO cells (EKO) and A431 cells pre-treated with E-cadherin blocking antibody (AbE). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to A431 cells after application of a force pulse. (c) Bead detachment data in CAFs transfected with siRNA Control (CT) and CAF-siNcad (siN). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to CAFs after application of a force pulse. (d) Illustration of the magnetic twisting experimental setup. (e) Representative fluorescence (top) and bright field (bottom) images showing the recruitment of β-catenin, P-cadherin, and E-cadherin in A431 cells subjected to magnetic stimulation using N-cadherin-coated magnetic beads. Yellow asterisks indicate the location of the beads. Scale bars, 5µm. (f) Quantification of the recruitment of β-catenin, P-cadherin and N-cadherin mediated by N-cadherin coated beads with/without (+/- Force) mechanical stimulation. (g) Representative bead traces for A431 cells and CAFs in response to a series of force pulses applied to beads coated with N-cadherin (red), E-cadherin (blue), P-cadherin (green) or uncoated (black). Vertical bars, 200nm. (h) Stiffening of the A431 cell-bead contact defined as the time evolution of the ratio between applied force and bead displacement relative to baseline (N-,E-,P-cadherin coated beads, and uncoated beads). (i) Stiffening of the CAF-bead contact. (j) Stiffening of the cell/E-cadherin-coated bead contact for control A431 cells (A431-WT) and α-catenin mutants. (k) Stiffening of the cell/N-cadherin-coated bead contact for A431-WT cells and α-catenin mutants. (l) for sample numbers and statistical analysis. Error bars represent s.e.m.

    Journal: Nature cell biology

    Article Title: A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

    doi: 10.1038/ncb3478

    Figure Lengend Snippet: Heterophilic E-cadherin/N-cadherin junctions withstand forces and trigger mechanotransduction (a) Illustration of the magnetic tweezers experimental setup. (b) Bead detachment data in A431 cells (CT), A431-EcadKO cells (EKO) and A431 cells pre-treated with E-cadherin blocking antibody (AbE). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to A431 cells after application of a force pulse. (c) Bead detachment data in CAFs transfected with siRNA Control (CT) and CAF-siNcad (siN). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to CAFs after application of a force pulse. (d) Illustration of the magnetic twisting experimental setup. (e) Representative fluorescence (top) and bright field (bottom) images showing the recruitment of β-catenin, P-cadherin, and E-cadherin in A431 cells subjected to magnetic stimulation using N-cadherin-coated magnetic beads. Yellow asterisks indicate the location of the beads. Scale bars, 5µm. (f) Quantification of the recruitment of β-catenin, P-cadherin and N-cadherin mediated by N-cadherin coated beads with/without (+/- Force) mechanical stimulation. (g) Representative bead traces for A431 cells and CAFs in response to a series of force pulses applied to beads coated with N-cadherin (red), E-cadherin (blue), P-cadherin (green) or uncoated (black). Vertical bars, 200nm. (h) Stiffening of the A431 cell-bead contact defined as the time evolution of the ratio between applied force and bead displacement relative to baseline (N-,E-,P-cadherin coated beads, and uncoated beads). (i) Stiffening of the CAF-bead contact. (j) Stiffening of the cell/E-cadherin-coated bead contact for control A431 cells (A431-WT) and α-catenin mutants. (k) Stiffening of the cell/N-cadherin-coated bead contact for A431-WT cells and α-catenin mutants. (l) for sample numbers and statistical analysis. Error bars represent s.e.m.

    Article Snippet: To visualize N-cadherin in CAFs and E-cadherin in A431 cells during time lapse experiments, unless stated otherwise cells were transfected with the N-cadherin-EGFP Plasmid (Addgene, #18870) and E-cadherin Ruby Plasmid (from Kurt Anderson’s lab) respectively two days before experiments using the Neon transfection device according to the manufacturer’s instructions (Invitrogen).

    Techniques: Blocking Assay, Transfection, Fluorescence, Magnetic Beads