vimentin Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher vimentin
    Ovarian fibrosarcoma pathology. ( A ) Hematoxylin-Eosin staining at 40x, ( B ) 100x and ( C ) 200x. ( D ) Immunohistochemistry of <t>Vimentin.</t> ( E ) Immunohistochemistry of Inhibin. Scale bars are shown at the right bottom of each micrograph.
    Vimentin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Thermo Fisher
    Average 99 stars, based on 2690 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    93
    Millipore vimentin
    Distribution of <t>Vimentin</t> or Trypanosoma cruzi antigen on control or infected cells and parasites. A and B) Uninfected LLC MK2 cells reacted to antivimentin abs(A) or anti-T.cruzi abs(B). C and D) T.cruzi infected LLC MK2 cells reacted to antivimentin abs(C) or anti-T.cruzi abs(D). E and F) T.cruzi promastigote forms from in vitro culture reacted to antivimentin abs (E) or anti-T.cruzi abs(F). Cells, infected cells or parasites forms were reacted with Anti Vimentin mAb or chronic infection chagasic serum and revealed with adequate conjugate (x1000) (see Methods).
    Vimentin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4029 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Millipore
    Average 93 stars, based on 4029 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc vimentin
    Knockdown of circKIF4A inhibits proliferation and metastasis of TNBC a si-circKIF4A #2 successfully knocked down circKIF4A. b A CCK-8 assay to detect cell proliferation. c A colony formation assay to detect cell colony-forming ability. d Colony formation number was quantified by ImageJ. e A Transwell assay to assess cell migratory ability. f The number of invasive cells was quantified by ImageJ. g A wound-healing assay to assess cell migratory capability. h Wound closure was quantified by ImageJ. i-j Immunofluorescence staining of E-cadherin and <t>vimentin.</t> k Xenograft models were established. l Summary of tumor weights. m Representative image of luciferase signals of lung metastatic nodules. n Representative images of lung metastatic nodules and HE-stained sections. o The number of metastatic nodules was quantified. ** P
    Vimentin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 9169 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Abcam vimentin
    The effects of βKlotho overexpression in PC3 cells. PC3 cells were transfected with either EV or βKlotho. (A) A CCK-8 assay, (B) a colony formation assay, (C) an apoptosis assay and (D) Transwell migration and invasion assays were performed at the indicated times. (E) PC3-EV and PC3-βKlotho cells were treated with 3% CSS for 24 h, then the cells were treated with or without 10 nM of DHT for 48 h. Western blotting was performed to detect the expression of βKlotho, E-cadherin, N-cadherin, <t>vimentin,</t> p-ERK1/2 and ERK1/2. GAPDH was used as the loading control. (F) The morphology of PC3 cells transfected with either EV or βKlotho. The cells were observed using phase contrast microscopy at ×200 magnification. Scale bar, 50 µm; **P
    Vimentin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 7726 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Abcam
    Average 99 stars, based on 7726 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology vimentin
    EMT tumor cells are resistant to CTX treatment both in vitro and in vivo a, b , Long term CTX treatment in vitro results in GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 µM). Fluorescent imaging (a) and flow cytometry (quantified, b, n=3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to control untreated cells c, d , EMT status of lung nodules in competitive survival assay. Representative fluorescent images of Tri-PyMT lung metastases in untreated control lungs ( c ) and CTX-treated lungs ( d ), depicting RFP+ and GFP+ tumor cells. Immunostaining showing E-cadherin (E-cad), <t>vimentin</t> (Vim) in white pseudocolor. White arrow indicates GFP+ tumor cells with epithelial phenotypes (E-cad+/Vim−), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad−/Vim+). Nuclei were counter stained with DAPI. n =5.
    Vimentin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 5601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Santa Cruz Biotechnology
    Average 93 stars, based on 5601 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    94
    Agilent technologies vimentin
    Effect of <t>anti-vimentin</t> antibody on flow-dependent platelet adhesion to collagen. Whole blood containing sheep IgG molecule or sheep anti-human vimentin was perfused over surfaces coated with collagen fibrils and analyzed as described in . The
    Vimentin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 4893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Agilent technologies
    Average 94 stars, based on 4893 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    Abcam anti vimentin
    Adiponectin promotes E-cadherin expression and downregulates <t>vimentin</t> expression in NSCLC cells. (A) Western blot analysis demonstrating the expression levels of E-cadherin and vimentin following incubation of cells with adiponectin. (A-D) Exogenous adiponectin promoted overexpression of E-cadherin and underexpression of vimentin in NSCLC cells as revealed by western blotting. GAPDH served as a loading control, *P
    Anti Vimentin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti vimentin/product/Abcam
    Average 99 stars, based on 2496 article reviews
    Price from $9.99 to $1999.99
    anti vimentin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti vimentin antibody
    Adiponectin promotes E-cadherin expression and downregulates <t>vimentin</t> expression in NSCLC cells. (A) Western blot analysis demonstrating the expression levels of E-cadherin and vimentin following incubation of cells with adiponectin. (A-D) Exogenous adiponectin promoted overexpression of E-cadherin and underexpression of vimentin in NSCLC cells as revealed by western blotting. GAPDH served as a loading control, *P
    Monoclonal Anti Vimentin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti vimentin antibody/product/Millipore
    Average 99 stars, based on 1840 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti vimentin antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Becton Dickinson vimentin
    EMT tumor cells are resistant to CTX treatment both in vitro and in vivo a, b , Long term CTX treatment in vitro results in GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 µM). Fluorescent imaging (a) and flow cytometry (quantified, b, n=3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to control untreated cells c, d , EMT status of lung nodules in competitive survival assay. Representative fluorescent images of Tri-PyMT lung metastases in untreated control lungs ( c ) and CTX-treated lungs ( d ), depicting RFP+ and GFP+ tumor cells. Immunostaining showing E-cadherin (E-cad), <t>vimentin</t> (Vim) in white pseudocolor. White arrow indicates GFP+ tumor cells with epithelial phenotypes (E-cad+/Vim−), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad−/Vim+). Nuclei were counter stained with DAPI. n =5.
    Vimentin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Becton Dickinson
    Average 94 stars, based on 1315 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    Millipore anti vimentin
    Immunocytochemical staining of GS-1 cells using antibodies against (a) cytokeratin, (b) <t>vimentin,</t> (c) fibronectin, and (d) desmin (green). The cell nuclei were counterstained with diaminophenylindole (DAPI; blue) (bar=100 µ m).
    Anti Vimentin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti vimentin/product/Millipore
    Average 99 stars, based on 1314 article reviews
    Price from $9.99 to $1999.99
    anti vimentin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Abcam anti vimentin antibody epr3776 cytoskeleton marker
    Immunocytochemical staining of GS-1 cells using antibodies against (a) cytokeratin, (b) <t>vimentin,</t> (c) fibronectin, and (d) desmin (green). The cell nuclei were counterstained with diaminophenylindole (DAPI; blue) (bar=100 µ m).
    Anti Vimentin Antibody Epr3776 Cytoskeleton Marker, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti vimentin antibody epr3776 cytoskeleton marker/product/Abcam
    Average 94 stars, based on 354 article reviews
    Price from $9.99 to $1999.99
    anti vimentin antibody epr3776 cytoskeleton marker - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    Proteintech rabbit vimentin polyclonal
    Immunocytochemical staining of GS-1 cells using antibodies against (a) cytokeratin, (b) <t>vimentin,</t> (c) fibronectin, and (d) desmin (green). The cell nuclei were counterstained with diaminophenylindole (DAPI; blue) (bar=100 µ m).
    Rabbit Vimentin Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit vimentin polyclonal/product/Proteintech
    Average 99 stars, based on 333 article reviews
    Price from $9.99 to $1999.99
    rabbit vimentin polyclonal - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher gene exp vim hs00185584 m1
    Effect of STS knockdown in T24 cells. (A) Two siRNAs for STS (si-STS A and si-STS B) reduced mRNA expression of STS in T24 cells. Relative STS expression levels are presented normalized to those in N cells. (B) Effect of siRNA knockdown on cell proliferation was evaluated using T24 cells. Each cell count was normalized to that of the N group. No significant difference was observed between si-STS-transfected and N cells. Effect of siRNA knockdown on cell migration and invasion ability was also evaluated using T24 cells. si-STS A- and B-transfected cells exhibited significantly reduced (C) migration and (D) invasion abilities compared with N cells. Scale bar indicates 500 µm. (E and F) Reverse transcription-quantitative polymerase chain reaction analyses of T24 cells transfected with siRNAs. Each gene expression level was normalized to that in N cells. (E) E-cadherin was upregulated and (F) <t>vimentin</t> was significantly downregulated by STS knockdown. *P
    Gene Exp Vim Hs00185584 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp vim hs00185584 m1/product/Thermo Fisher
    Average 99 stars, based on 547 article reviews
    Price from $9.99 to $1999.99
    gene exp vim hs00185584 m1 - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    93
    Agilent technologies mouse anti vimentin
    Prolonged respiratory HDM exposure induces epithelial-to-mesenchymal transition. Lung sections (15 µm thick) were prepared from control mice and mice exposed to HDM for 5, 10 or 15 weeks and immunofluorescent staining for the co-expression of the LacZ reporter in airway epithelial cells with α-SMA and occludin (A–D) and with <t>vimentin</t> and E-cadherin (E–H) was performed. Scale bars 10 µm. Quantification of lung fibrosis was performed by morphometric analysis of lung sections stained for α-SMA (I) or LacZ and vimentin (J). * p
    Mouse Anti Vimentin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti vimentin/product/Agilent technologies
    Average 93 stars, based on 517 article reviews
    Price from $9.99 to $1999.99
    mouse anti vimentin - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    99
    Millipore anti vimentin antibody
    Models of epithelial and mesenchymal cells. Images of vehicle (10 mM citric acid)-treated and TGFβ1-treated (20 ng/ml for 4 days) A549 cells ( a ), MCF-7 cells ( c ) and BEAS-2B ( e ) cells. Images of A549 ( b ) and MCF-7 ( d ) cultured in the presence of 10% or 1% FBS for 4 days. Images of serum-supplemented (+10% FBS) and serum-starved (-FBS) (bottom) BEAS-2B cells ( f ) after 7 days of serum starvation. Western blot analysis of β-actin, GAPDH, E-cadherin, N-cadherin and <t>Vimentin</t> in the three cell model cell lines ( d–f ). N-cadherin and Vimentin were not detectable in MCF-7 and BEAS-2B cells.
    Anti Vimentin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti vimentin antibody/product/Millipore
    Average 99 stars, based on 416 article reviews
    Price from $9.99 to $1999.99
    anti vimentin antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Epitomics vimentin
    Analysis of <t>vimentin</t> distribution in analyzed cells. A) Vimentin was detected by anti-vimentin at the cell surface of HT22 cells transfected with siRNA for 48 h at 37°C by FACS analysis. B) Confocal microscopy of vimentin in HT22 cells transfected for 48 h at 37°C. The green (oregon green 488) anti-vimentin, DNA staining in blue (Dapi), rhodamine red-staining for actin and a merge image are shown for each panel. In the enlarged images the cell boundaries are shown. Significant difference between the vimentin distribution was detected for the transfected cells (lower panel) in comparison to the control (upper panel). Scale bar = 20 µM. C) Detection of vimentin at the cell surface of J774A.1 cells transfected with siRNA. D) Confocal microscopy of vimentin distribution in J774A.1 cells transfected for 48 h.
    Vimentin, supplied by Epitomics, used in various techniques. Bioz Stars score: 94/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vimentin/product/Epitomics
    Average 94 stars, based on 467 article reviews
    Price from $9.99 to $1999.99
    vimentin - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    Image Search Results


    Ovarian fibrosarcoma pathology. ( A ) Hematoxylin-Eosin staining at 40x, ( B ) 100x and ( C ) 200x. ( D ) Immunohistochemistry of Vimentin. ( E ) Immunohistochemistry of Inhibin. Scale bars are shown at the right bottom of each micrograph.

    Journal: Scientific Reports

    Article Title: Genomics of a pediatric ovarian fibrosarcoma. Association with the DICER1 syndrome

    doi: 10.1038/s41598-018-21663-9

    Figure Lengend Snippet: Ovarian fibrosarcoma pathology. ( A ) Hematoxylin-Eosin staining at 40x, ( B ) 100x and ( C ) 200x. ( D ) Immunohistochemistry of Vimentin. ( E ) Immunohistochemistry of Inhibin. Scale bars are shown at the right bottom of each micrograph.

    Article Snippet: Immunohistochemistry was performed against Vimentin (MA5-11883, Invitrogen, CA, USA; 1:100 dilution) and Inhibin (5692, Bio SB, CA, USA; 1:100 dilution) as reported previously .

    Techniques: Staining, Immunohistochemistry

    Distribution of Vimentin or Trypanosoma cruzi antigen on control or infected cells and parasites. A and B) Uninfected LLC MK2 cells reacted to antivimentin abs(A) or anti-T.cruzi abs(B). C and D) T.cruzi infected LLC MK2 cells reacted to antivimentin abs(C) or anti-T.cruzi abs(D). E and F) T.cruzi promastigote forms from in vitro culture reacted to antivimentin abs (E) or anti-T.cruzi abs(F). Cells, infected cells or parasites forms were reacted with Anti Vimentin mAb or chronic infection chagasic serum and revealed with adequate conjugate (x1000) (see Methods).

    Journal: Arquivos Brasileiros de Cardiologia

    Article Title: Vimentin and Anti Vimentin Antibodies in Chagas' Disease

    doi: 10.5935/abc.20180038

    Figure Lengend Snippet: Distribution of Vimentin or Trypanosoma cruzi antigen on control or infected cells and parasites. A and B) Uninfected LLC MK2 cells reacted to antivimentin abs(A) or anti-T.cruzi abs(B). C and D) T.cruzi infected LLC MK2 cells reacted to antivimentin abs(C) or anti-T.cruzi abs(D). E and F) T.cruzi promastigote forms from in vitro culture reacted to antivimentin abs (E) or anti-T.cruzi abs(F). Cells, infected cells or parasites forms were reacted with Anti Vimentin mAb or chronic infection chagasic serum and revealed with adequate conjugate (x1000) (see Methods).

    Article Snippet: Monoclonal mouse Anti-Vimentin antibody (V6630) and vimentin from bovine lens was obtained commercially (Sigma Aldrich, Saint Louis, Missouri, USA).

    Techniques: Infection, In Vitro

    Knockdown of circKIF4A inhibits proliferation and metastasis of TNBC a si-circKIF4A #2 successfully knocked down circKIF4A. b A CCK-8 assay to detect cell proliferation. c A colony formation assay to detect cell colony-forming ability. d Colony formation number was quantified by ImageJ. e A Transwell assay to assess cell migratory ability. f The number of invasive cells was quantified by ImageJ. g A wound-healing assay to assess cell migratory capability. h Wound closure was quantified by ImageJ. i-j Immunofluorescence staining of E-cadherin and vimentin. k Xenograft models were established. l Summary of tumor weights. m Representative image of luciferase signals of lung metastatic nodules. n Representative images of lung metastatic nodules and HE-stained sections. o The number of metastatic nodules was quantified. ** P

    Journal: Molecular Cancer

    Article Title: circKIF4A acts as a prognostic factor and mediator to regulate the progression of triple-negative breast cancer

    doi: 10.1186/s12943-019-0946-x

    Figure Lengend Snippet: Knockdown of circKIF4A inhibits proliferation and metastasis of TNBC a si-circKIF4A #2 successfully knocked down circKIF4A. b A CCK-8 assay to detect cell proliferation. c A colony formation assay to detect cell colony-forming ability. d Colony formation number was quantified by ImageJ. e A Transwell assay to assess cell migratory ability. f The number of invasive cells was quantified by ImageJ. g A wound-healing assay to assess cell migratory capability. h Wound closure was quantified by ImageJ. i-j Immunofluorescence staining of E-cadherin and vimentin. k Xenograft models were established. l Summary of tumor weights. m Representative image of luciferase signals of lung metastatic nodules. n Representative images of lung metastatic nodules and HE-stained sections. o The number of metastatic nodules was quantified. ** P

    Article Snippet: Immunofluorescence staining Cells were seeded and fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.5% Triton X-100 for 10 min and blocked with 4% bovine serum albumin for 1 h then incubated with primary antibodies of E-cadherin and vimentin (Cell Signaling Technology, USA, 1:100) at 4 °C overnight.

    Techniques: CCK-8 Assay, Colony Assay, Transwell Assay, Wound Healing Assay, Immunofluorescence, Staining, Luciferase

    circKIF4A acts as a sponge for miR-375 a The expression levels of nuclear control (18S), cytoplasmic control (GAPDH) and circKIF4A were detected. b The predicted binding sites of miR-375 within the circKIF4A sequence. c The miR-375 expression in TNBC cell lines. d Luciferase assay of cells cotransfected with miR-375 mimics and wild type or mutant luciferase reporter. e MS2-based RIP assay transfected with MS2bs-circKIF4A, MS2bs-circKIF4Amt or control. f A CCK-8 assay to detect cell proliferation. g A colony formation assay to detect cell colony-forming ability. h The colony formation number was quantified by ImageJ. i A Transwell assay to assess cell migration ability. j The number of invasive was quantified by ImageJ. k A wound-healing assay to detect cell migration ability. l Wound closure was quantified by ImageJ. m-n Immunofluorescence staining for E-cadherin and vimentin. o Xenograft models were established. p Summary of the tumor weights. q Representative image of luciferase signals of lung metastatic nodules. r Representative images of lung metastatic nodules and HE-stained sections. s The number of metastatic nodules was quantified. ** P

    Journal: Molecular Cancer

    Article Title: circKIF4A acts as a prognostic factor and mediator to regulate the progression of triple-negative breast cancer

    doi: 10.1186/s12943-019-0946-x

    Figure Lengend Snippet: circKIF4A acts as a sponge for miR-375 a The expression levels of nuclear control (18S), cytoplasmic control (GAPDH) and circKIF4A were detected. b The predicted binding sites of miR-375 within the circKIF4A sequence. c The miR-375 expression in TNBC cell lines. d Luciferase assay of cells cotransfected with miR-375 mimics and wild type or mutant luciferase reporter. e MS2-based RIP assay transfected with MS2bs-circKIF4A, MS2bs-circKIF4Amt or control. f A CCK-8 assay to detect cell proliferation. g A colony formation assay to detect cell colony-forming ability. h The colony formation number was quantified by ImageJ. i A Transwell assay to assess cell migration ability. j The number of invasive was quantified by ImageJ. k A wound-healing assay to detect cell migration ability. l Wound closure was quantified by ImageJ. m-n Immunofluorescence staining for E-cadherin and vimentin. o Xenograft models were established. p Summary of the tumor weights. q Representative image of luciferase signals of lung metastatic nodules. r Representative images of lung metastatic nodules and HE-stained sections. s The number of metastatic nodules was quantified. ** P

    Article Snippet: Immunofluorescence staining Cells were seeded and fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.5% Triton X-100 for 10 min and blocked with 4% bovine serum albumin for 1 h then incubated with primary antibodies of E-cadherin and vimentin (Cell Signaling Technology, USA, 1:100) at 4 °C overnight.

    Techniques: Expressing, Binding Assay, Sequencing, Luciferase, Mutagenesis, Transfection, CCK-8 Assay, Colony Assay, Transwell Assay, Migration, Wound Healing Assay, Immunofluorescence, Staining

    The mesenchymal phenotype was maintained in eribulin-resistant MDA-MB-231 and Hs578T cells The expression of pSmad2, ZEB1, E-cadherin, vimentin, and Slug was analyzed by western blotting using eribulin-resistant MDA-MB-231 and Hs578T cells and the respective parental cells. Cell growth and migration ability were investigated using cell growth and wound healing assays, respectively. ( A ) Representative results of western blot analyses in eribulin-resistant MDA-MB-231 and Hs578T cells (E) and their parental cells (P). β-Actin was used as a loading control. ( B ) Results of cell growth assays performed using eribulin-resistant MDA-MB-231 and Hs578T cells (E) and their parental cells (P). Error bars represent standard deviation; experiments were performed in triplicate. ( C ) Results of wound healing assays performed in eribulin-resistant MDA-MB-231 and Hs578T cells (E) and their parental cells (P). Representative images of wound healing assays after a confluent cell monolayer was scratched at 0 and 24 h for MDA-MB-231 cells and 0 and 12 h for Hs578T cells (upper panels, scale bar = 200 µm). The average wound closure of 100 randomized points was calculated as a ratio of the initial wound size (lower panels). Error bars represent standard deviation based on three independent experiments. * p

    Journal: Oncotarget

    Article Title: Combination of two anti-tubulin agents, eribulin and paclitaxel, enhances anti-tumor effects on triple-negative breast cancer through mesenchymal-epithelial transition

    doi: 10.18632/oncotarget.25184

    Figure Lengend Snippet: The mesenchymal phenotype was maintained in eribulin-resistant MDA-MB-231 and Hs578T cells The expression of pSmad2, ZEB1, E-cadherin, vimentin, and Slug was analyzed by western blotting using eribulin-resistant MDA-MB-231 and Hs578T cells and the respective parental cells. Cell growth and migration ability were investigated using cell growth and wound healing assays, respectively. ( A ) Representative results of western blot analyses in eribulin-resistant MDA-MB-231 and Hs578T cells (E) and their parental cells (P). β-Actin was used as a loading control. ( B ) Results of cell growth assays performed using eribulin-resistant MDA-MB-231 and Hs578T cells (E) and their parental cells (P). Error bars represent standard deviation; experiments were performed in triplicate. ( C ) Results of wound healing assays performed in eribulin-resistant MDA-MB-231 and Hs578T cells (E) and their parental cells (P). Representative images of wound healing assays after a confluent cell monolayer was scratched at 0 and 24 h for MDA-MB-231 cells and 0 and 12 h for Hs578T cells (upper panels, scale bar = 200 µm). The average wound closure of 100 randomized points was calculated as a ratio of the initial wound size (lower panels). Error bars represent standard deviation based on three independent experiments. * p

    Article Snippet: The following antibodies and dilutions were used: anti-E-cadherin (1:500, Gene Tax) and anti-vimentin (1:100; Cell Signaling Technology).

    Techniques: Multiple Displacement Amplification, Expressing, Western Blot, Migration, Standard Deviation

    FOXQ1 is essential for TAM-induced EMT in GC cells. (A and B) The expression of mesenchymal marker vimentin was decreased, while the epithelial marker E-cadherin was increased in MKN45-FOXQ1-shRNA cells co-cultured with THP-1 cells. (C and D) The expression of mesenchymal marker vimentin was decreased, while the epithelial marker E-cadherin was increased in MKN74-FOXQ1-shRNA cells co-cultured with THP-1 cells. Values are depicted as the mean ± SD; *P

    Journal: Oncology Reports

    Article Title: Tumor-associated macrophages induce the expression of FOXQ1 to promote epithelial-mesenchymal transition and metastasis in gastric cancer cells

    doi: 10.3892/or.2017.5877

    Figure Lengend Snippet: FOXQ1 is essential for TAM-induced EMT in GC cells. (A and B) The expression of mesenchymal marker vimentin was decreased, while the epithelial marker E-cadherin was increased in MKN45-FOXQ1-shRNA cells co-cultured with THP-1 cells. (C and D) The expression of mesenchymal marker vimentin was decreased, while the epithelial marker E-cadherin was increased in MKN74-FOXQ1-shRNA cells co-cultured with THP-1 cells. Values are depicted as the mean ± SD; *P

    Article Snippet: Rabbit anti-FOXQ1 (1:100; ab51340; Abcam, Cambridge, MA, USA), rabbit anti-E-cadherin (1:100; AF0131; Affinity, Sterling, VA, USA), rabbit anti-vimentin (1:100; 5741; Cell Signaling Technology, Inc., Beverly, MA, USA) and mouse anti-β-actin (1:100; T0022; Affinity) were used as primary antibodies.

    Techniques: Expressing, Marker, shRNA, Cell Culture

    The effects of βKlotho overexpression in PC3 cells. PC3 cells were transfected with either EV or βKlotho. (A) A CCK-8 assay, (B) a colony formation assay, (C) an apoptosis assay and (D) Transwell migration and invasion assays were performed at the indicated times. (E) PC3-EV and PC3-βKlotho cells were treated with 3% CSS for 24 h, then the cells were treated with or without 10 nM of DHT for 48 h. Western blotting was performed to detect the expression of βKlotho, E-cadherin, N-cadherin, vimentin, p-ERK1/2 and ERK1/2. GAPDH was used as the loading control. (F) The morphology of PC3 cells transfected with either EV or βKlotho. The cells were observed using phase contrast microscopy at ×200 magnification. Scale bar, 50 µm; **P

    Journal: Oncology Reports

    Article Title: βKlotho inhibits androgen/androgen receptor-associated epithelial-mesenchymal transition in prostate cancer through inactivation of ERK1/2 signaling

    doi: 10.3892/or.2018.6399

    Figure Lengend Snippet: The effects of βKlotho overexpression in PC3 cells. PC3 cells were transfected with either EV or βKlotho. (A) A CCK-8 assay, (B) a colony formation assay, (C) an apoptosis assay and (D) Transwell migration and invasion assays were performed at the indicated times. (E) PC3-EV and PC3-βKlotho cells were treated with 3% CSS for 24 h, then the cells were treated with or without 10 nM of DHT for 48 h. Western blotting was performed to detect the expression of βKlotho, E-cadherin, N-cadherin, vimentin, p-ERK1/2 and ERK1/2. GAPDH was used as the loading control. (F) The morphology of PC3 cells transfected with either EV or βKlotho. The cells were observed using phase contrast microscopy at ×200 magnification. Scale bar, 50 µm; **P

    Article Snippet: The anti-E-cadherin (1:1,000 for western blot analysis; 1:200 for IHC; cat. no. ab1416), N-cadherin (1:1,000 for western blot analysis, 1:200 for IHC; cat. no. ab98952), vimentin (1:1,000 for western blot analysis; cat. no. ab8978) and βKlotho antibodies (1:1,000 for western blot analysis; 1:100 for IHC; cat. no. ab106794) were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Over Expression, Transfection, CCK-8 Assay, Colony Assay, Apoptosis Assay, Migration, Western Blot, Expressing, Microscopy

    The effects of βKlotho knockdown in LNCaP cells. LNCaP cells were transfected with either control siRNA or βKlotho siRNA, followed by (A) a CCK-8 assay, (B) an apoptosis assay and (C) Transwell migration and invasion assays that were performed at the indicated times. (D) LNCaP cells were transfected with either control siRNA or βKlotho siRNA for 48 h, and western blotting was performed to detect the expression of βKlotho, E-cadherin, N-cadherin and vimentin. GAPDH was used as the loading control. (E) The morphology of LNCaP cells transfected with either control siRNA or βKlotho siRNA for 48 h. The cells were observed using phase contrast microscopy at ×200 magnification. Scale bar, 50 µm; * P

    Journal: Oncology Reports

    Article Title: βKlotho inhibits androgen/androgen receptor-associated epithelial-mesenchymal transition in prostate cancer through inactivation of ERK1/2 signaling

    doi: 10.3892/or.2018.6399

    Figure Lengend Snippet: The effects of βKlotho knockdown in LNCaP cells. LNCaP cells were transfected with either control siRNA or βKlotho siRNA, followed by (A) a CCK-8 assay, (B) an apoptosis assay and (C) Transwell migration and invasion assays that were performed at the indicated times. (D) LNCaP cells were transfected with either control siRNA or βKlotho siRNA for 48 h, and western blotting was performed to detect the expression of βKlotho, E-cadherin, N-cadherin and vimentin. GAPDH was used as the loading control. (E) The morphology of LNCaP cells transfected with either control siRNA or βKlotho siRNA for 48 h. The cells were observed using phase contrast microscopy at ×200 magnification. Scale bar, 50 µm; * P

    Article Snippet: The anti-E-cadherin (1:1,000 for western blot analysis; 1:200 for IHC; cat. no. ab1416), N-cadherin (1:1,000 for western blot analysis, 1:200 for IHC; cat. no. ab98952), vimentin (1:1,000 for western blot analysis; cat. no. ab8978) and βKlotho antibodies (1:1,000 for western blot analysis; 1:100 for IHC; cat. no. ab106794) were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Transfection, CCK-8 Assay, Apoptosis Assay, Migration, Western Blot, Expressing, Microscopy

    PDLSCs culture and identification (( A ) single PDLSC; ( B ) A clone of the cyanine blue dye PDLSCs; ( C ) PDLSCs Polyclonal formation; ( D ) PDLSCs lipid droplet formed after lipid induction, oil red O staining positive; ( E ) mineralized nodules formed after PDLSCs were osteo-induced, and Alizarin Red staining was positive; ( F ) RT-PCR after mineralized induction, PDLSCs expressed osteogenic proteins (COL1, OCN, RUNX-2, BSP); ( G, H ) Flow cytometry detection of the mesenchymal stem cell marker of PDLSCs was tested positive for STRO-1 and CD146; ( I, J ) Cultured cells were identified by immunofluorescence technology staining using anti-vimentin monoclonal antibody and cytokeratin polyclonal antibody.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Receptor-Interacting Protein 3/Caspase-8 May Regulate Inflammatory Response and Promote Tissue Regeneration in the Periodontal Microenvironment

    doi: 10.12659/MSM.909192

    Figure Lengend Snippet: PDLSCs culture and identification (( A ) single PDLSC; ( B ) A clone of the cyanine blue dye PDLSCs; ( C ) PDLSCs Polyclonal formation; ( D ) PDLSCs lipid droplet formed after lipid induction, oil red O staining positive; ( E ) mineralized nodules formed after PDLSCs were osteo-induced, and Alizarin Red staining was positive; ( F ) RT-PCR after mineralized induction, PDLSCs expressed osteogenic proteins (COL1, OCN, RUNX-2, BSP); ( G, H ) Flow cytometry detection of the mesenchymal stem cell marker of PDLSCs was tested positive for STRO-1 and CD146; ( I, J ) Cultured cells were identified by immunofluorescence technology staining using anti-vimentin monoclonal antibody and cytokeratin polyclonal antibody.

    Article Snippet: Anti-vimentin antibody (ab8978), Anti-pan Cytokeratin antibody (ab7753), and Alexa Fluor 647(ab150171) were bought from Abcam and Goat Anti-Mouse IgG and Cy3 was bought from Zhuangzhibio (Xi’an, China).

    Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Marker, Cell Culture, Immunofluorescence

    EMT tumor cells are resistant to CTX treatment both in vitro and in vivo a, b , Long term CTX treatment in vitro results in GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 µM). Fluorescent imaging (a) and flow cytometry (quantified, b, n=3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to control untreated cells c, d , EMT status of lung nodules in competitive survival assay. Representative fluorescent images of Tri-PyMT lung metastases in untreated control lungs ( c ) and CTX-treated lungs ( d ), depicting RFP+ and GFP+ tumor cells. Immunostaining showing E-cadherin (E-cad), vimentin (Vim) in white pseudocolor. White arrow indicates GFP+ tumor cells with epithelial phenotypes (E-cad+/Vim−), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad−/Vim+). Nuclei were counter stained with DAPI. n =5.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: EMT tumor cells are resistant to CTX treatment both in vitro and in vivo a, b , Long term CTX treatment in vitro results in GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 µM). Fluorescent imaging (a) and flow cytometry (quantified, b, n=3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to control untreated cells c, d , EMT status of lung nodules in competitive survival assay. Representative fluorescent images of Tri-PyMT lung metastases in untreated control lungs ( c ) and CTX-treated lungs ( d ), depicting RFP+ and GFP+ tumor cells. Immunostaining showing E-cadherin (E-cad), vimentin (Vim) in white pseudocolor. White arrow indicates GFP+ tumor cells with epithelial phenotypes (E-cad+/Vim−), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad−/Vim+). Nuclei were counter stained with DAPI. n =5.

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques: In Vitro, In Vivo, Imaging, Flow Cytometry, Cytometry, Clonogenic Cell Survival Assay, Immunostaining, Staining

    Characterization of the primary tumor and lung metastasis of Tri-PyMT/Vim mice Tri-PyMT/Vim mice were obtained by crossing MMTV-PyMT, Vimentin-Cre and Rosa26-GRFP transgenic mice. Sections of primary tumors (a) and lungs (b) were immunostained for PyMT, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of the primary tumor and lung metastasis of Tri-PyMT/Vim mice Tri-PyMT/Vim mice were obtained by crossing MMTV-PyMT, Vimentin-Cre and Rosa26-GRFP transgenic mice. Sections of primary tumors (a) and lungs (b) were immunostained for PyMT, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques: Mouse Assay, Transgenic Assay

    Characterization of EMT status of orthotopic Tri-Vim-PyMT primary tumor Sections of Tri-Vim-PyMT orthotopic primary tumors ( a ) and metastatic lung ( b ) were immunostained for E-cadherin and vimentin (in white psuedocolor). As expected, RFP+ tumor cells are entirely E-cadherin positive and vimentin negative, GFP+ tumor cells are vimentin positive and E-cadherin negative, and lung metastases are epithelial and RFP+.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of EMT status of orthotopic Tri-Vim-PyMT primary tumor Sections of Tri-Vim-PyMT orthotopic primary tumors ( a ) and metastatic lung ( b ) were immunostained for E-cadherin and vimentin (in white psuedocolor). As expected, RFP+ tumor cells are entirely E-cadherin positive and vimentin negative, GFP+ tumor cells are vimentin positive and E-cadherin negative, and lung metastases are epithelial and RFP+.

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques:

    Characterization of the primary tumor and lung metastasis of Tri-Neu mice Sections of primary tumors (left panel) and lungs (right panel) from Tri-Neu mice were immunostained for CD45, Neu, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of the primary tumor and lung metastasis of Tri-Neu mice Sections of primary tumors (left panel) and lungs (right panel) from Tri-Neu mice were immunostained for CD45, Neu, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques: Mouse Assay

    The EMT lineage tracing system reports EMT in tumor cells with high fidelity a , Scatter plots from flow cytometry analysis of Tri-PyMT primary tumor cells, depicting GFP+ and RFP+ populations in the primary tumor immediately after sorting of RFP+ cells (P1), and after 10 passages in culture with 10% FBS (P10+10%FBS). Numbers indicate the percentage of RFP+ and GFP+ cells in the total population. b , Phase contrast/fluorescent overlay image of Tri-PyMT cells in culture. c , Western blot of sorted RFP+ and GFP+ Tri-PyMT cells for E-cadherin, vimentin and β-actin as a loading control. Representative of 2 individual experiments. For gel source data, see Supplementary Figure 1 . d , Representative imaging of GFP+ and RFP+ tumor cells in primary tumors (PT) and lung metastases (LM) in the orthotopic model (n=8 mice). Arrow indicates scattered GFP+EMT tumor cells in the primary tumor. e , Q-RT-PCR analysis of relative expression of EMT markers in RFP+ and GFP+ cells sorted from orthotopic Tri-PyMT primary tumors. GAPDH served as the internal control. Data are reported as mean ± SEM, n=4 primary tumors.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: The EMT lineage tracing system reports EMT in tumor cells with high fidelity a , Scatter plots from flow cytometry analysis of Tri-PyMT primary tumor cells, depicting GFP+ and RFP+ populations in the primary tumor immediately after sorting of RFP+ cells (P1), and after 10 passages in culture with 10% FBS (P10+10%FBS). Numbers indicate the percentage of RFP+ and GFP+ cells in the total population. b , Phase contrast/fluorescent overlay image of Tri-PyMT cells in culture. c , Western blot of sorted RFP+ and GFP+ Tri-PyMT cells for E-cadherin, vimentin and β-actin as a loading control. Representative of 2 individual experiments. For gel source data, see Supplementary Figure 1 . d , Representative imaging of GFP+ and RFP+ tumor cells in primary tumors (PT) and lung metastases (LM) in the orthotopic model (n=8 mice). Arrow indicates scattered GFP+EMT tumor cells in the primary tumor. e , Q-RT-PCR analysis of relative expression of EMT markers in RFP+ and GFP+ cells sorted from orthotopic Tri-PyMT primary tumors. GAPDH served as the internal control. Data are reported as mean ± SEM, n=4 primary tumors.

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques: Flow Cytometry, Cytometry, Western Blot, Imaging, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

    Establishing an orthotopic model with sorted RFP+ Tri-PyMT cells a , Flow cytometry plots show Tri-PyMT cells before and after sorting for RFP+ cells. Numbers indicate the percentage and purity of RFP+ cells used for establishing orthotopic breast tumors in mice. b , Schematic of the orthotopic breast tumor model with sorted RFP+ Tri-PyMT cells. Cells are injected into the mammary gland of wild type mice to generate primary breast tumors, resection of primary tumor at 4 weeks and lung metastases evaluation in another 4 weeks. c , Characterization of tumor cells in the primary tumor, disseminated tumor cells (DTCs) and tumor cells in the lung metastasis of the Tri-PyMT orthotopic model. Sections of primary tumors and lungs from Tri-PyMT orthotopic mice were immunostained for E-cadherin and Vimentin (in white pseudocolor). Essentially all RFP+ tumor cells are detected as E-cad+/Vim−, while the scattered GFP+ tumor cells in the primary tumor are E-Cad−/Vim+ (as indicated by arrows in the top panel). Representative images are shown (n=8). d, Plot shows the percentage of GFP+ cells out of total tumor cells (GFP+ plus RFP+, n=6).

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Establishing an orthotopic model with sorted RFP+ Tri-PyMT cells a , Flow cytometry plots show Tri-PyMT cells before and after sorting for RFP+ cells. Numbers indicate the percentage and purity of RFP+ cells used for establishing orthotopic breast tumors in mice. b , Schematic of the orthotopic breast tumor model with sorted RFP+ Tri-PyMT cells. Cells are injected into the mammary gland of wild type mice to generate primary breast tumors, resection of primary tumor at 4 weeks and lung metastases evaluation in another 4 weeks. c , Characterization of tumor cells in the primary tumor, disseminated tumor cells (DTCs) and tumor cells in the lung metastasis of the Tri-PyMT orthotopic model. Sections of primary tumors and lungs from Tri-PyMT orthotopic mice were immunostained for E-cadherin and Vimentin (in white pseudocolor). Essentially all RFP+ tumor cells are detected as E-cad+/Vim−, while the scattered GFP+ tumor cells in the primary tumor are E-Cad−/Vim+ (as indicated by arrows in the top panel). Representative images are shown (n=8). d, Plot shows the percentage of GFP+ cells out of total tumor cells (GFP+ plus RFP+, n=6).

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Injection

    Characterization of the primary tumor and lung metastasis of Tri-PyMT mice Sections of primary tumors (a) and lungs (b) from Tri-PyMT mice were immunostained for E-cadherin (E-cad, top), vimentin (Vim, middle) and CD45 (bottom) in white psuedocolor. Representative images are shown (n > 5 mice). Note the co-localization of PyMT with RFP, and CD45 with GFP (as indicated by arrows) in both primary tumors and lung metastases.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of the primary tumor and lung metastasis of Tri-PyMT mice Sections of primary tumors (a) and lungs (b) from Tri-PyMT mice were immunostained for E-cadherin (E-cad, top), vimentin (Vim, middle) and CD45 (bottom) in white psuedocolor. Representative images are shown (n > 5 mice). Note the co-localization of PyMT with RFP, and CD45 with GFP (as indicated by arrows) in both primary tumors and lung metastases.

    Article Snippet: Primary antibodies used in this study include CD45 (30-F11, BioLegend), E-cadherin (DECMA-1, BioLegend), vimentin (sc-7557, Santa Cruz), PyMT (ab15085, Abcam), Neu (sc-284, Santa Cruz), Ki67 (ab15580, Abcam), and Active Caspase-3 (C92–605, BD Pharmingen).

    Techniques: Mouse Assay

    Vimentin phosphorylation is decreased in Rab7a-silenced cells. (A–B) Total extracts of Hela cells treated with control RNA (scr) or with Rab7a siRNA, as indicated, were subjected to Western blot analysis using anti-phospho Ser38 (A) or Ser55 (B), anti-tubulin and anti-Rab7a. (C) Total extracts of Hela cells treated with control RNA (scr) or with Rab7a siRNA and transfected with HA-Rab7a, as indicated, were subjected to Western blot analysis using anti-phospho Ser38 and anti-phospho Ser55 vimentins, anti-Rab7a, anti-HA and anti-α-tubulin antibodies.

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: Vimentin phosphorylation is decreased in Rab7a-silenced cells. (A–B) Total extracts of Hela cells treated with control RNA (scr) or with Rab7a siRNA, as indicated, were subjected to Western blot analysis using anti-phospho Ser38 (A) or Ser55 (B), anti-tubulin and anti-Rab7a. (C) Total extracts of Hela cells treated with control RNA (scr) or with Rab7a siRNA and transfected with HA-Rab7a, as indicated, were subjected to Western blot analysis using anti-phospho Ser38 and anti-phospho Ser55 vimentins, anti-Rab7a, anti-HA and anti-α-tubulin antibodies.

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Western Blot, Transfection

    Rab7a is able to interact with the phosphorylated form of vimentin. (A) Total extract of HeLa cells expressing HA-tagged Rab7a wt or Rab7a V162M were subjected to immunoprecipitation using anti-HA resin. Western blot analysis of immunoprecipitates was made using anti-HA, anti-phospho-vimentins Ser55 and Ser 38. (B) Soluble and insoluble fractions of Hela cells expressing HA-tagged Rab7a wt or Rab7a V162M were subjected to Western blot analysis using anti-phospho-vimentins Ser55 and Ser 38. (C) Soluble and insoluble fractions of HeLa cells expressing HA-tagged Rab7a wt or Rab7a V162M were immunoprecipitated using anti-HA resin. Immunoprecipitates were subjected to WB analysis using anti-phospho-vimentins Ser38 and Ser55, anti-vimentin and anti-HA antibodies.

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: Rab7a is able to interact with the phosphorylated form of vimentin. (A) Total extract of HeLa cells expressing HA-tagged Rab7a wt or Rab7a V162M were subjected to immunoprecipitation using anti-HA resin. Western blot analysis of immunoprecipitates was made using anti-HA, anti-phospho-vimentins Ser55 and Ser 38. (B) Soluble and insoluble fractions of Hela cells expressing HA-tagged Rab7a wt or Rab7a V162M were subjected to Western blot analysis using anti-phospho-vimentins Ser55 and Ser 38. (C) Soluble and insoluble fractions of HeLa cells expressing HA-tagged Rab7a wt or Rab7a V162M were immunoprecipitated using anti-HA resin. Immunoprecipitates were subjected to WB analysis using anti-phospho-vimentins Ser38 and Ser55, anti-vimentin and anti-HA antibodies.

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Immunoprecipitation, Western Blot

    Rab7a overexpression induces vimentin phosphorylation. Total extracts of control HeLa cells (NT) or HeLa cells transfected with HA-tagged Rab7a wt and mutant proteins, as indicated, were subjected to Western blot analysis using anti-phospho-vimentin Ser38 (A) or Ser55 (C), anti-tubulin and anti-HA antibodies. (B, D) Quantification of phosphorylated vimentin Ser38 (B) or Ser55 (D) in cells expressing Rab7a wt and mutant proteins. Values represent the mean mean ± s.d. of four independent experiments. Intensities of the bands were quantified by densitometry, normalized against the amount of tubulin and plotted relatively to the values obtained in control HeLa cells (NT). Values obtained in cells expressing disease-causing Rab7a mutant proteins were found to be significantly different from the values obtained in control (NT) cells (*p

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: Rab7a overexpression induces vimentin phosphorylation. Total extracts of control HeLa cells (NT) or HeLa cells transfected with HA-tagged Rab7a wt and mutant proteins, as indicated, were subjected to Western blot analysis using anti-phospho-vimentin Ser38 (A) or Ser55 (C), anti-tubulin and anti-HA antibodies. (B, D) Quantification of phosphorylated vimentin Ser38 (B) or Ser55 (D) in cells expressing Rab7a wt and mutant proteins. Values represent the mean mean ± s.d. of four independent experiments. Intensities of the bands were quantified by densitometry, normalized against the amount of tubulin and plotted relatively to the values obtained in control HeLa cells (NT). Values obtained in cells expressing disease-causing Rab7a mutant proteins were found to be significantly different from the values obtained in control (NT) cells (*p

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Over Expression, Transfection, Mutagenesis, Western Blot, Expressing

    Rab7a interacts directly with vimentin. AH109 yeast cells were co-transformed with pGADT7-vimentin and pGBKT7, PGBKT7-Rab7b, PGBKT7-Rab9 or pGBKT7-Rab7a wt or mutant constructs. (A) Double transformants were plated on WL and HWL synthetic medium and grown at 30 °C for two days. (B) The β-galactosidase activity of double transformants was measured using o -nitrophenyl-β- d -galactoside as substrate as detailed in Materials and methods . Activities are measured as arbitrary relative units and represent mean ± s.d. values of 6 independent transformants from three independent experiments. (C) NiNTA resin alone or together with purified His-Rab7 was incubated with total extracts of HeLa cells overexpressing HA-vimentin. After affinity chromatography proteins were subjected to Western blot analysis using anti-HA and anti-Rab7a antibodies. (D) Glutathione resin alone or together with purified GST or GST-vimentin was incubated with purified His-Rab7a. After affinity chromatography proteins were subjected to Western blot analysis using anti-GST and anti-Rab7a antibodies. (E) Total extracts of HeLa cells and immunoprecipitates obtained with no antibodies (no ab), with mouse anti-Rab7a and with mouse IgG, as indicated, were subjected to WB analysis using rabbit anti-Rab7a and rabbit anti-vimentin antibodies.

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: Rab7a interacts directly with vimentin. AH109 yeast cells were co-transformed with pGADT7-vimentin and pGBKT7, PGBKT7-Rab7b, PGBKT7-Rab9 or pGBKT7-Rab7a wt or mutant constructs. (A) Double transformants were plated on WL and HWL synthetic medium and grown at 30 °C for two days. (B) The β-galactosidase activity of double transformants was measured using o -nitrophenyl-β- d -galactoside as substrate as detailed in Materials and methods . Activities are measured as arbitrary relative units and represent mean ± s.d. values of 6 independent transformants from three independent experiments. (C) NiNTA resin alone or together with purified His-Rab7 was incubated with total extracts of HeLa cells overexpressing HA-vimentin. After affinity chromatography proteins were subjected to Western blot analysis using anti-HA and anti-Rab7a antibodies. (D) Glutathione resin alone or together with purified GST or GST-vimentin was incubated with purified His-Rab7a. After affinity chromatography proteins were subjected to Western blot analysis using anti-GST and anti-Rab7a antibodies. (E) Total extracts of HeLa cells and immunoprecipitates obtained with no antibodies (no ab), with mouse anti-Rab7a and with mouse IgG, as indicated, were subjected to WB analysis using rabbit anti-Rab7a and rabbit anti-vimentin antibodies.

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Transformation Assay, Mutagenesis, Construct, Activity Assay, Purification, Incubation, Affinity Chromatography, Western Blot

    Intracellular localization of Rab7a and vimentin filaments. HeLa cells overexpressing GFP-tagged Rab7a wt (A–B) or L129F (C), K157N (D), N161T (E) V162M and (F) mutant proteins were subjected to immunofluorescence analysis using an anti-vimentin antibody followed by a Cy3 secondary antibody. (G–H) Hela cells were subjected to immunofluorescence analysis using anti-Rab7a and anti-vimentin antibodies followed by Cy2 and a Cy3 conjugated secondary antibodies, respectively. The images represent maximum-intensity projections of Z stacks. Bar = 10 μm.

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: Intracellular localization of Rab7a and vimentin filaments. HeLa cells overexpressing GFP-tagged Rab7a wt (A–B) or L129F (C), K157N (D), N161T (E) V162M and (F) mutant proteins were subjected to immunofluorescence analysis using an anti-vimentin antibody followed by a Cy3 secondary antibody. (G–H) Hela cells were subjected to immunofluorescence analysis using anti-Rab7a and anti-vimentin antibodies followed by Cy2 and a Cy3 conjugated secondary antibodies, respectively. The images represent maximum-intensity projections of Z stacks. Bar = 10 μm.

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Mutagenesis, Immunofluorescence

    CMT2B-causing Rab7a mutant proteins show altered binding to vimentin. (A) HA-tagged Rab7a wt and mutant proteins were expressed in HeLa cells and immunoprecipitated using an anti-HA resin. Immunoprecipitates were subjected to Western blot analysis using anti-vimentin and anti-HA antibodies. (B) Quantification of four independent co-immunoprecipitation experiments. Intensities of the bands were quantified by densitometry, normalized against the amount of the Rab7a protein expressed. Values are mean ± s.d. (C) Purified His-tagged Rab7a wt and mutant proteins, as indicated, were incubated with total extracts of HeLa cells. Precipitated proteins after affinity chromatography with NiNTA resin were loaded on SDS-PAGE and subjected to Western blot analysis using anti-vimentin and anti-Rab7a antibodies. (D) Quantification of four independent pull-down experiments. Intensities of the bands were quantified by densitometry and normalized against the amount of the Rab7a protein. Values are mean ± s.d. Values obtained expressing disease-causing Rab7a mutant proteins were found to be significantly different from the values obtained in cells expressing Rab7a wt (*p

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: CMT2B-causing Rab7a mutant proteins show altered binding to vimentin. (A) HA-tagged Rab7a wt and mutant proteins were expressed in HeLa cells and immunoprecipitated using an anti-HA resin. Immunoprecipitates were subjected to Western blot analysis using anti-vimentin and anti-HA antibodies. (B) Quantification of four independent co-immunoprecipitation experiments. Intensities of the bands were quantified by densitometry, normalized against the amount of the Rab7a protein expressed. Values are mean ± s.d. (C) Purified His-tagged Rab7a wt and mutant proteins, as indicated, were incubated with total extracts of HeLa cells. Precipitated proteins after affinity chromatography with NiNTA resin were loaded on SDS-PAGE and subjected to Western blot analysis using anti-vimentin and anti-Rab7a antibodies. (D) Quantification of four independent pull-down experiments. Intensities of the bands were quantified by densitometry and normalized against the amount of the Rab7a protein. Values are mean ± s.d. Values obtained expressing disease-causing Rab7a mutant proteins were found to be significantly different from the values obtained in cells expressing Rab7a wt (*p

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, Western Blot, Purification, Incubation, Affinity Chromatography, SDS Page, Expressing

    Modulation of Rab7a expression alters vimentin filament organization. (A) Total extract of Hela cells (NT) or of HeLa cells expressing HA-tagged Rab7a wt and mutant proteins as indicated were subjected to Western blot analysis with anti-vimentin, anti tubulin and anti-HA antibodies. Total extracts (B) or soluble and insoluble extracts (C) of Hela cells treated with control RNA (scr) or with Rab7a siRNA as indicated were subjected to Western blot analysis using anti-Rab7a, anti-tubulin and anti-vimentin antibodies. (D) Quantification of soluble and insoluble vimentins in Rab7a-depleted HeLa cells. The values represent the mean ± s.d. of four independent experiments. The intensities were quantified by densitometry, normalized against the amount of tubulin and plotted relatively to the intensities obtained in cells transfected with control RNA (scr). Values obtained in Rab7a-depleted cells were found to be significantly different from the values obtained in control (scr) cells (**p

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: Modulation of Rab7a expression alters vimentin filament organization. (A) Total extract of Hela cells (NT) or of HeLa cells expressing HA-tagged Rab7a wt and mutant proteins as indicated were subjected to Western blot analysis with anti-vimentin, anti tubulin and anti-HA antibodies. Total extracts (B) or soluble and insoluble extracts (C) of Hela cells treated with control RNA (scr) or with Rab7a siRNA as indicated were subjected to Western blot analysis using anti-Rab7a, anti-tubulin and anti-vimentin antibodies. (D) Quantification of soluble and insoluble vimentins in Rab7a-depleted HeLa cells. The values represent the mean ± s.d. of four independent experiments. The intensities were quantified by densitometry, normalized against the amount of tubulin and plotted relatively to the intensities obtained in cells transfected with control RNA (scr). Values obtained in Rab7a-depleted cells were found to be significantly different from the values obtained in control (scr) cells (**p

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Mutagenesis, Western Blot, Transfection

    A model for the role of Rab7a in vimentin assembly. Rab7a induces vimentin phosphorylation at different sites and this, in turn, causes vimentin disassembly. Rab7a is able to bind both to the soluble and filamentous forms of vimentin.

    Journal: Biochimica et Biophysica Acta

    Article Title: Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a

    doi: 10.1016/j.bbamcr.2013.02.024

    Figure Lengend Snippet: A model for the role of Rab7a in vimentin assembly. Rab7a induces vimentin phosphorylation at different sites and this, in turn, causes vimentin disassembly. Rab7a is able to bind both to the soluble and filamentous forms of vimentin.

    Article Snippet: 2.6 Antibodies Mouse monoclonal 9E10 anti-Myc (ab32) and rabbit polyclonal anti-HA (ab9110) antibodies were from Abcam (Cambridge, UK), rabbit polyclonal anti-Rab7a (R4779), mouse monoclonal anti-Rab7a (R8779) and mouse monoclonal anti-tubulin (T5168) were from Sigma-Aldrich (St. Louis, MO), while anti-vimentin (sc-6260) and anti-vimentin phospho Ser38 (sc-16673) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques:

    Ajoene Inhibits the Invasion and Migration of MDA-MB-231 and HeLa Cells. Wound healing assay: Following introduction of a scratch wound into the cell layer of ( a and c ) MDA-MB-231 or ( b and d ) HeLa cells; native cells, or cells silenced for vimentin expression were incubated with 10 μM ZA in DMSO or DMSO alone for 24 h. Migration into the wound was then quantified using Image J software. Transwell invasion and migration assay: ( e ) MDA-MB-231 cells or ( f ) HeLa cells were transfected with vimentin siRNA and treated as described above for 24 h. The ability of cells to invade and migrate through the matrigel membrane was quantified by counting the crystal violet stained cells, data displayed as mean ± SD. The results of a single representative experiment is shown; however experiments were performed in duplicate

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Ajoene Inhibits the Invasion and Migration of MDA-MB-231 and HeLa Cells. Wound healing assay: Following introduction of a scratch wound into the cell layer of ( a and c ) MDA-MB-231 or ( b and d ) HeLa cells; native cells, or cells silenced for vimentin expression were incubated with 10 μM ZA in DMSO or DMSO alone for 24 h. Migration into the wound was then quantified using Image J software. Transwell invasion and migration assay: ( e ) MDA-MB-231 cells or ( f ) HeLa cells were transfected with vimentin siRNA and treated as described above for 24 h. The ability of cells to invade and migrate through the matrigel membrane was quantified by counting the crystal violet stained cells, data displayed as mean ± SD. The results of a single representative experiment is shown; however experiments were performed in duplicate

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Migration, Multiple Displacement Amplification, Wound Healing Assay, Expressing, Incubation, Software, Transfection, Staining

    Ajoene Rescues the Enhanced Migratory Potential of Vimentin Overexpressing Cells. ( a ) Vimentin was transiently overexpressed using human vimentin cDNA cloned into pCMV3, in both HeLa and MDA-MB-231 cells as shown and quantified by immunoblot. A scratch wound was then introduced into MDA-MB-231 ( b and d ) or HeLa ( c or e ) cells and 10 μM ZA in DMSO or DMSO alone was incubated with the cells for 24 h. Migration into the wound was then quantified using Image J software. The results of a single representative experiment is shown; however experiments were performed in duplicate

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Ajoene Rescues the Enhanced Migratory Potential of Vimentin Overexpressing Cells. ( a ) Vimentin was transiently overexpressed using human vimentin cDNA cloned into pCMV3, in both HeLa and MDA-MB-231 cells as shown and quantified by immunoblot. A scratch wound was then introduced into MDA-MB-231 ( b and d ) or HeLa ( c or e ) cells and 10 μM ZA in DMSO or DMSO alone was incubated with the cells for 24 h. Migration into the wound was then quantified using Image J software. The results of a single representative experiment is shown; however experiments were performed in duplicate

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Clone Assay, Multiple Displacement Amplification, Incubation, Migration, Software

    Computational modelling of the Vimentin tetramer showing the environment of Cys-328. The structure of the vimentin tetramer (PDBID 3KLT) was chosen, prepared and modelled using Schrödinger software. The structure of the tetramer is labelled and shown using a space filling representation for chain ( a ) (grey) and modified cartoon representation for chains ( b , c , d ) (cyan, yellow and magenta), The four cysteine thiols are coloured yellow and are exposed at the termini of the tetramer. The environment for the cysteine of each chain in the tetramer is illustrated. The thiol hydrogen points in the direction of the glutamate in chain ( a ), towards the carbonyl oxygen in chains ( c , d ) and towards Gln-324 in chain ( b )

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Computational modelling of the Vimentin tetramer showing the environment of Cys-328. The structure of the vimentin tetramer (PDBID 3KLT) was chosen, prepared and modelled using Schrödinger software. The structure of the tetramer is labelled and shown using a space filling representation for chain ( a ) (grey) and modified cartoon representation for chains ( b , c , d ) (cyan, yellow and magenta), The four cysteine thiols are coloured yellow and are exposed at the termini of the tetramer. The environment for the cysteine of each chain in the tetramer is illustrated. The thiol hydrogen points in the direction of the glutamate in chain ( a ), towards the carbonyl oxygen in chains ( c , d ) and towards Gln-324 in chain ( b )

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Software, Modification

    Purification and Identification of Vimentin from DP-treated MDA-MB-231 cells. Proposed disulfide exchange reaction occurring between a cysteine sulfhydryl group on a target protein with ( a ) Z-ajoene (ZA) or its analogue ( b ) dansyl-ajoene (DP). ( c ) Lysate collected from MDA-MB-231 breast cancer cells treated with 25 μM ZA or DP show many dansyl-labelled proteins by immunoblot when probed with an anti-dansyl primary antibody in the DP-treated sample only. The experiment was performed under non-reducing conditions. ( d ) Separation of the dansyl-labelled proteins in MDA-MB-231 cell lysate by 2D gel electrophoresis under non-reducing conditions. A predominant band (circled) was observed in the immunoblot which was excised from the corresponding gel and identified by MALDI-TOF MS/MS to be vimentin

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Purification and Identification of Vimentin from DP-treated MDA-MB-231 cells. Proposed disulfide exchange reaction occurring between a cysteine sulfhydryl group on a target protein with ( a ) Z-ajoene (ZA) or its analogue ( b ) dansyl-ajoene (DP). ( c ) Lysate collected from MDA-MB-231 breast cancer cells treated with 25 μM ZA or DP show many dansyl-labelled proteins by immunoblot when probed with an anti-dansyl primary antibody in the DP-treated sample only. The experiment was performed under non-reducing conditions. ( d ) Separation of the dansyl-labelled proteins in MDA-MB-231 cell lysate by 2D gel electrophoresis under non-reducing conditions. A predominant band (circled) was observed in the immunoblot which was excised from the corresponding gel and identified by MALDI-TOF MS/MS to be vimentin

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Purification, Multiple Displacement Amplification, Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry

    Validation of Vimentin as an Ajoene Target. ( a ) Immunoblot of human recombinant vimentin treated with DP (100 μM) in the absence or presence of DTT (100 mM), probed with a primary anti-dansyl and anti-vimentin (H-84) antibody. ( b ) Human recombinant vimentin was treated with 100 μM ZA or DP and purified by SDS-PAGE. The band excised from the gel was digested with trypsin and fragments were identified by MS/MS MALDI-TOFF mass spectrometry. The Cys-328 containing fragment qvqsltcevdalk was detected in the control and treated samples carrying a 2+ charge where m/z = [M + 2H]2+. ( c ) In the samples treated with ZA or DP, the predicted m/z ratio of the modified fragment was observed

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Validation of Vimentin as an Ajoene Target. ( a ) Immunoblot of human recombinant vimentin treated with DP (100 μM) in the absence or presence of DTT (100 mM), probed with a primary anti-dansyl and anti-vimentin (H-84) antibody. ( b ) Human recombinant vimentin was treated with 100 μM ZA or DP and purified by SDS-PAGE. The band excised from the gel was digested with trypsin and fragments were identified by MS/MS MALDI-TOFF mass spectrometry. The Cys-328 containing fragment qvqsltcevdalk was detected in the control and treated samples carrying a 2+ charge where m/z = [M + 2H]2+. ( c ) In the samples treated with ZA or DP, the predicted m/z ratio of the modified fragment was observed

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Recombinant, Purification, SDS Page, Mass Spectrometry, Modification

    Ajoene Induces Increased Expression of Vimentin. MDA-MB-231 (top) or HeLa (bottom) cells were treated with either DMSO (control) or 10 μM ZA in DMSO up to 8 h. Proteins collected from the cell lysate were separated by SDS-PAGE and vimentin expression was quantified by immunoblot probed with a primary anti-vimentin antibody (V9). The blots shown are a representative experiment of two independent determinations

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Ajoene Induces Increased Expression of Vimentin. MDA-MB-231 (top) or HeLa (bottom) cells were treated with either DMSO (control) or 10 μM ZA in DMSO up to 8 h. Proteins collected from the cell lysate were separated by SDS-PAGE and vimentin expression was quantified by immunoblot probed with a primary anti-vimentin antibody (V9). The blots shown are a representative experiment of two independent determinations

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Expressing, Multiple Displacement Amplification, SDS Page

    Ajoene Disrupts the Vimentin Filament Network in MDA-MB-231 and HeLa cells. 40x Phase contrast images of MDA-MB-231 ( a ) or HeLa ( d ) cells treated with DMSO (control) or 10 μM ZA in DMSO for 24 h. Cell viability assay: MDA-MB-231 ( b ) or HeLa ( e ) cells treated with DMSO (control) or with ZA (0, 5, 10, 20 or 40 μM) for 6 h or 24 h. Immunofluorescence: MDA-MB-231 ( c ) or HeLa ( f ) cells treated with 20 μM ZA for 6 h, then fixed and immunostained with vimentin primary antibodies (V9, H84 or V4630). Control cells treated with DMSO alone. Images obtained by confocal scanning laser microscopy

    Journal: BMC Cancer

    Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

    doi: 10.1186/s12885-019-5388-8

    Figure Lengend Snippet: Ajoene Disrupts the Vimentin Filament Network in MDA-MB-231 and HeLa cells. 40x Phase contrast images of MDA-MB-231 ( a ) or HeLa ( d ) cells treated with DMSO (control) or 10 μM ZA in DMSO for 24 h. Cell viability assay: MDA-MB-231 ( b ) or HeLa ( e ) cells treated with DMSO (control) or with ZA (0, 5, 10, 20 or 40 μM) for 6 h or 24 h. Immunofluorescence: MDA-MB-231 ( c ) or HeLa ( f ) cells treated with 20 μM ZA for 6 h, then fixed and immunostained with vimentin primary antibodies (V9, H84 or V4630). Control cells treated with DMSO alone. Images obtained by confocal scanning laser microscopy

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-vimentin (V9) (1:1000, Santa Cruz for V9 and H84 and Sigma-Aldrich for V4630), anti-GAPDH (1:1000, Santa Cruz).

    Techniques: Multiple Displacement Amplification, Viability Assay, Immunofluorescence, Microscopy

    Effect of anti-vimentin antibody on flow-dependent platelet adhesion to collagen. Whole blood containing sheep IgG molecule or sheep anti-human vimentin was perfused over surfaces coated with collagen fibrils and analyzed as described in . The

    Journal: Blood

    Article Title: Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

    doi: 10.1182/blood-2013-10-530428

    Figure Lengend Snippet: Effect of anti-vimentin antibody on flow-dependent platelet adhesion to collagen. Whole blood containing sheep IgG molecule or sheep anti-human vimentin was perfused over surfaces coated with collagen fibrils and analyzed as described in . The

    Article Snippet: VWF and A1A2A3 proteins bound to vimentin were detected by enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-VWF-HRP conjugate (Dako), whereas the antibody VP-1 was used for the A2 protein, followed by a secondary goat anti-mouse IgG-HRP conjugate (Pierce).

    Techniques: Flow Cytometry

    Interaction of the A1A2A3 proteins with vimentin. (A) WT-A1A2A3 protein or gain-of-function A1A2A3 mutant (250 nmol/L) was incubated with immobilized vimentin in the absence of ristocetin. The bound protein was determined by ELISA, using the antibody

    Journal: Blood

    Article Title: Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

    doi: 10.1182/blood-2013-10-530428

    Figure Lengend Snippet: Interaction of the A1A2A3 proteins with vimentin. (A) WT-A1A2A3 protein or gain-of-function A1A2A3 mutant (250 nmol/L) was incubated with immobilized vimentin in the absence of ristocetin. The bound protein was determined by ELISA, using the antibody

    Article Snippet: VWF and A1A2A3 proteins bound to vimentin were detected by enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-VWF-HRP conjugate (Dako), whereas the antibody VP-1 was used for the A2 protein, followed by a secondary goat anti-mouse IgG-HRP conjugate (Pierce).

    Techniques: Mutagenesis, Incubation, Enzyme-linked Immunosorbent Assay

    Effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3 protein. Whole blood from human or mice expressing human (h)GPIbα in platelets was mixed with sheep IgG molecule (negative control) or sheep anti-vim antibody (100

    Journal: Blood

    Article Title: Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

    doi: 10.1182/blood-2013-10-530428

    Figure Lengend Snippet: Effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3 protein. Whole blood from human or mice expressing human (h)GPIbα in platelets was mixed with sheep IgG molecule (negative control) or sheep anti-vim antibody (100

    Article Snippet: VWF and A1A2A3 proteins bound to vimentin were detected by enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-VWF-HRP conjugate (Dako), whereas the antibody VP-1 was used for the A2 protein, followed by a secondary goat anti-mouse IgG-HRP conjugate (Pierce).

    Techniques: Flow Cytometry, Mouse Assay, Expressing, Negative Control

    Effect of anti-vimentin antibody on flow-dependent platelet adhesion to fibrin(ogen). Whole blood containing sheep IgG molecule or sheep anti-vim antibody was mixed with ristocetin (0.5 mg/mL) and perfused over surfaces coated with fibrin(ogen) at a shear

    Journal: Blood

    Article Title: Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

    doi: 10.1182/blood-2013-10-530428

    Figure Lengend Snippet: Effect of anti-vimentin antibody on flow-dependent platelet adhesion to fibrin(ogen). Whole blood containing sheep IgG molecule or sheep anti-vim antibody was mixed with ristocetin (0.5 mg/mL) and perfused over surfaces coated with fibrin(ogen) at a shear

    Article Snippet: VWF and A1A2A3 proteins bound to vimentin were detected by enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-VWF-HRP conjugate (Dako), whereas the antibody VP-1 was used for the A2 protein, followed by a secondary goat anti-mouse IgG-HRP conjugate (Pierce).

    Techniques: Flow Cytometry

    Vimentin-deficient platelets have a reduced platelet adhesion to collagen and VWF. Whole blood from WT (n = 6) or vimentin knockout mice (n = 7) was perfused over surfaces coated with (A) collagen fibrils or (B) purified plasma murine VWF at a shear rate

    Journal: Blood

    Article Title: Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

    doi: 10.1182/blood-2013-10-530428

    Figure Lengend Snippet: Vimentin-deficient platelets have a reduced platelet adhesion to collagen and VWF. Whole blood from WT (n = 6) or vimentin knockout mice (n = 7) was perfused over surfaces coated with (A) collagen fibrils or (B) purified plasma murine VWF at a shear rate

    Article Snippet: VWF and A1A2A3 proteins bound to vimentin were detected by enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-VWF-HRP conjugate (Dako), whereas the antibody VP-1 was used for the A2 protein, followed by a secondary goat anti-mouse IgG-HRP conjugate (Pierce).

    Techniques: Knock-Out, Mouse Assay, Purification

    VWF binds to immobilized human vimentin. (A) Increasing concentrations of purified plasma VWF were mixed with ristocetin (0.2 mmol/L) (♦) or buffer (▪) and incubated with immobilized vimentin. Bound VWF was determined by ELISA using the

    Journal: Blood

    Article Title: Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

    doi: 10.1182/blood-2013-10-530428

    Figure Lengend Snippet: VWF binds to immobilized human vimentin. (A) Increasing concentrations of purified plasma VWF were mixed with ristocetin (0.2 mmol/L) (♦) or buffer (▪) and incubated with immobilized vimentin. Bound VWF was determined by ELISA using the

    Article Snippet: VWF and A1A2A3 proteins bound to vimentin were detected by enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-VWF-HRP conjugate (Dako), whereas the antibody VP-1 was used for the A2 protein, followed by a secondary goat anti-mouse IgG-HRP conjugate (Pierce).

    Techniques: Purification, Incubation, Enzyme-linked Immunosorbent Assay

    IHC expression of CK AE1/AE3 and vimentin in serous cystadenoma ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in serous cystadenoma ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    IHC expression of CK AE1/AE3 and vimentin in benign mucinous tumor of ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in benign mucinous tumor of ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    IHC expression of CK AE1/AE3 and vimentin in borderline serous tumor of ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in borderline serous tumor of ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    IHC expression of CK AE1/AE3 and vimentin in normal ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in normal ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    IHC expression of CK AE1/AE3 and vimentin in malignant mucinous tumor of ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in malignant mucinous tumor of ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    IHC expression of CK AE1/AE3 and vimentin in malignant serous tumor of ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in malignant serous tumor of ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    IHC expression of CK AE1/AE3 and vimentin in borderline mucinous tumor of ovary

    Journal: Iranian Journal of Pathology

    Article Title: Immunohistochemical Characterization of Normal Ovary and Common Epithelial Ovarian Neoplasm with a Monoclonal Antibody to Cytokeratin and Vimentin

    doi:

    Figure Lengend Snippet: IHC expression of CK AE1/AE3 and vimentin in borderline mucinous tumor of ovary

    Article Snippet: Sixty-six common epithelial ovarian tumors were studied using anti-cytokeratins (Monoclonal Mouse Anti-Human Cytokeratin Clones AE1/AE3; DAKO, Denmark,) and anti-vimentin (Monoclonal Mouse Anti-Vimentin, Clone V9; DAKO, Denmark,) to ascertain the intermediate filament profiles in formalin-fixed and paraffin-embedded surgical pathology materials.

    Techniques: Immunohistochemistry, Expressing

    Adiponectin promotes E-cadherin expression and downregulates vimentin expression in NSCLC cells. (A) Western blot analysis demonstrating the expression levels of E-cadherin and vimentin following incubation of cells with adiponectin. (A-D) Exogenous adiponectin promoted overexpression of E-cadherin and underexpression of vimentin in NSCLC cells as revealed by western blotting. GAPDH served as a loading control, *P

    Journal: Oncology Reports

    Article Title: Adiponectin inhibits migration and invasion by reversing epithelial-mesenchymal transition in non-small cell lung carcinoma

    doi: 10.3892/or.2018.6523

    Figure Lengend Snippet: Adiponectin promotes E-cadherin expression and downregulates vimentin expression in NSCLC cells. (A) Western blot analysis demonstrating the expression levels of E-cadherin and vimentin following incubation of cells with adiponectin. (A-D) Exogenous adiponectin promoted overexpression of E-cadherin and underexpression of vimentin in NSCLC cells as revealed by western blotting. GAPDH served as a loading control, *P

    Article Snippet: 4970 and 3700), goat anti-rabbit HRP (dilution 1:2,000; cat. no. 7074) and goat anti-mouse HRP (dilution 1:2,000; cat. no. 7076) (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-E-cadherin (dilution 1:2,000; cat. no. ab76055) and anti-vimentin (dilution 1:2,000; cat. no. ab92547) (Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Incubation, Over Expression

    Knockdown of Twist inhibits the migration and invasion of NSCLC cells. (A) Western blotting demonstrating that basal levels of E-cadherin expression were high in HCC827, moderate in A549 and low in NCI-H1299 cells while basal levels of vimentin expression were high in NCI-H1299, moderate in HCC827 and low in A549 cells, n=3 (each experiment). (B) Confocal microscopy images confirming the expression levels, n=3 (each experiment). Twist knockdown results in significant inhibition of (C-E) migration [ # P

    Journal: Oncology Reports

    Article Title: Adiponectin inhibits migration and invasion by reversing epithelial-mesenchymal transition in non-small cell lung carcinoma

    doi: 10.3892/or.2018.6523

    Figure Lengend Snippet: Knockdown of Twist inhibits the migration and invasion of NSCLC cells. (A) Western blotting demonstrating that basal levels of E-cadherin expression were high in HCC827, moderate in A549 and low in NCI-H1299 cells while basal levels of vimentin expression were high in NCI-H1299, moderate in HCC827 and low in A549 cells, n=3 (each experiment). (B) Confocal microscopy images confirming the expression levels, n=3 (each experiment). Twist knockdown results in significant inhibition of (C-E) migration [ # P

    Article Snippet: 4970 and 3700), goat anti-rabbit HRP (dilution 1:2,000; cat. no. 7074) and goat anti-mouse HRP (dilution 1:2,000; cat. no. 7076) (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-E-cadherin (dilution 1:2,000; cat. no. ab76055) and anti-vimentin (dilution 1:2,000; cat. no. ab92547) (Abcam, Cambridge, MA, USA).

    Techniques: Migration, Western Blot, Expressing, Confocal Microscopy, Inhibition

    Increased susceptibility to ZIKV infection in murine male GCs due to reduced IFN signaling. a , b Immunostaining ( a ) and quantification ( b ) of ZIKV-infected wild-type C57/BL6J (IFU = 1 × 10 5 ) and Ifnar1 −/− (IFU = 1 × 10 2 ) testis at 6 dpi. c Induction of ISGs in ZIKV-infected (MOI = 0.1 PFU per cell) GCs, SCs, LCs, and MCs at 72 hpi ( n = 3) measured by qRT-PCR. d , e Immunostaining (VIM for SCs and CALB2 for LCs) ( d ) and quantification ( n = 5 for each group) ( e ) of pSTAT1 (Tyr701) after 1 h of 1000 U ml −1 of IFNα. f Percent of infected GCs and Vero cells ( n = 3) measured by flow cytometry after a 24 h incubation with 1000 U ml −1 of IFNα followed by a 1 h incubation with ZIKV (MOI = 0.1 PFU per cell). Data are presented as mean ± s.e.m. and analyzed by two-sided t test and one-way ANOVA, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, or no significance (ns). Scale bar, 50 μm. ZIKV strain is MR 766

    Journal: Nature Communications

    Article Title: Male germ cells support long-term propagation of Zika virus

    doi: 10.1038/s41467-018-04444-w

    Figure Lengend Snippet: Increased susceptibility to ZIKV infection in murine male GCs due to reduced IFN signaling. a , b Immunostaining ( a ) and quantification ( b ) of ZIKV-infected wild-type C57/BL6J (IFU = 1 × 10 5 ) and Ifnar1 −/− (IFU = 1 × 10 2 ) testis at 6 dpi. c Induction of ISGs in ZIKV-infected (MOI = 0.1 PFU per cell) GCs, SCs, LCs, and MCs at 72 hpi ( n = 3) measured by qRT-PCR. d , e Immunostaining (VIM for SCs and CALB2 for LCs) ( d ) and quantification ( n = 5 for each group) ( e ) of pSTAT1 (Tyr701) after 1 h of 1000 U ml −1 of IFNα. f Percent of infected GCs and Vero cells ( n = 3) measured by flow cytometry after a 24 h incubation with 1000 U ml −1 of IFNα followed by a 1 h incubation with ZIKV (MOI = 0.1 PFU per cell). Data are presented as mean ± s.e.m. and analyzed by two-sided t test and one-way ANOVA, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, or no significance (ns). Scale bar, 50 μm. ZIKV strain is MR 766

    Article Snippet: The cells were washed with PBS, blocked with blocking buffer containing 0.3% Triton X in 5% horse serum for 30 min at room temperature, and incubated with primary antibodies including rabbit anti-DDX4 (1:500, Abcam, ab13840), rabbit anti-SOX9 (1:500, Millipore, AB5535), rabbit anti-vimentin (1:500, Abcam, ab92547 [EPR3776]), rabbit anti-17β-HSD (1:100, Proteintech, 13415-1-AP), rabbit anti-α-SMA (1:500, Millipore, ABT1487), rabbit anti-CALB2 (1:250, Novus Biologicals, NBP1-32244), rabbit anti-DAZL (1:500, Abcam, ab34139), mouse and rabbit anti-flavivirus group antigen (ZIKV-E) (1:500, Absolute, Ab00230-2.0 and Ab00230-23.0 [D1-4G2-4-15 (4G2)]), rabbit anti-pSTAT1 (Tyr701) (1:300, Cell Signaling, 9167 [58D6]), mouse IgG (1:500, Jackson ImmunoResearch, 015-000-003), and rabbit IgG (1:200, Jackson ImmunoResearch, 011-000-003) at 4 °C overnight.

    Techniques: Infection, Immunostaining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Incubation

    EMT tumor cells are resistant to CTX treatment both in vitro and in vivo a, b , Long term CTX treatment in vitro results in GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 µM). Fluorescent imaging (a) and flow cytometry (quantified, b, n=3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to control untreated cells c, d , EMT status of lung nodules in competitive survival assay. Representative fluorescent images of Tri-PyMT lung metastases in untreated control lungs ( c ) and CTX-treated lungs ( d ), depicting RFP+ and GFP+ tumor cells. Immunostaining showing E-cadherin (E-cad), vimentin (Vim) in white pseudocolor. White arrow indicates GFP+ tumor cells with epithelial phenotypes (E-cad+/Vim−), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad−/Vim+). Nuclei were counter stained with DAPI. n =5.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: EMT tumor cells are resistant to CTX treatment both in vitro and in vivo a, b , Long term CTX treatment in vitro results in GFP+ population. Tri-PyMT cells were subjected to 2 weeks cyclophosphamide (+CTX) treatment (4 µM). Fluorescent imaging (a) and flow cytometry (quantified, b, n=3) exhibit the percentage of GFP+ cells in the CTX-treated culture compared to control untreated cells c, d , EMT status of lung nodules in competitive survival assay. Representative fluorescent images of Tri-PyMT lung metastases in untreated control lungs ( c ) and CTX-treated lungs ( d ), depicting RFP+ and GFP+ tumor cells. Immunostaining showing E-cadherin (E-cad), vimentin (Vim) in white pseudocolor. White arrow indicates GFP+ tumor cells with epithelial phenotypes (E-cad+/Vim−), while the yellow arrow indicates GFP+ cells with mesenchymal phenotypes (E-cad−/Vim+). Nuclei were counter stained with DAPI. n =5.

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques: In Vitro, In Vivo, Imaging, Flow Cytometry, Cytometry, Clonogenic Cell Survival Assay, Immunostaining, Staining

    Characterization of the primary tumor and lung metastasis of Tri-PyMT/Vim mice Tri-PyMT/Vim mice were obtained by crossing MMTV-PyMT, Vimentin-Cre and Rosa26-GRFP transgenic mice. Sections of primary tumors (a) and lungs (b) were immunostained for PyMT, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of the primary tumor and lung metastasis of Tri-PyMT/Vim mice Tri-PyMT/Vim mice were obtained by crossing MMTV-PyMT, Vimentin-Cre and Rosa26-GRFP transgenic mice. Sections of primary tumors (a) and lungs (b) were immunostained for PyMT, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques: Mouse Assay, Transgenic Assay

    Characterization of EMT status of orthotopic Tri-Vim-PyMT primary tumor Sections of Tri-Vim-PyMT orthotopic primary tumors ( a ) and metastatic lung ( b ) were immunostained for E-cadherin and vimentin (in white psuedocolor). As expected, RFP+ tumor cells are entirely E-cadherin positive and vimentin negative, GFP+ tumor cells are vimentin positive and E-cadherin negative, and lung metastases are epithelial and RFP+.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of EMT status of orthotopic Tri-Vim-PyMT primary tumor Sections of Tri-Vim-PyMT orthotopic primary tumors ( a ) and metastatic lung ( b ) were immunostained for E-cadherin and vimentin (in white psuedocolor). As expected, RFP+ tumor cells are entirely E-cadherin positive and vimentin negative, GFP+ tumor cells are vimentin positive and E-cadherin negative, and lung metastases are epithelial and RFP+.

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques:

    Characterization of the primary tumor and lung metastasis of Tri-Neu mice Sections of primary tumors (left panel) and lungs (right panel) from Tri-Neu mice were immunostained for CD45, Neu, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of the primary tumor and lung metastasis of Tri-Neu mice Sections of primary tumors (left panel) and lungs (right panel) from Tri-Neu mice were immunostained for CD45, Neu, E-cadherin, and Vimentin (in white psuedocolor). Representative images are shown (n > 5 mice). Note that both primary tumors and lung metastases are largely composed of epithelial RFP+ tumor cells.

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques: Mouse Assay

    The EMT lineage tracing system reports EMT in tumor cells with high fidelity a , Scatter plots from flow cytometry analysis of Tri-PyMT primary tumor cells, depicting GFP+ and RFP+ populations in the primary tumor immediately after sorting of RFP+ cells (P1), and after 10 passages in culture with 10% FBS (P10+10%FBS). Numbers indicate the percentage of RFP+ and GFP+ cells in the total population. b , Phase contrast/fluorescent overlay image of Tri-PyMT cells in culture. c , Western blot of sorted RFP+ and GFP+ Tri-PyMT cells for E-cadherin, vimentin and β-actin as a loading control. Representative of 2 individual experiments. For gel source data, see Supplementary Figure 1 . d , Representative imaging of GFP+ and RFP+ tumor cells in primary tumors (PT) and lung metastases (LM) in the orthotopic model (n=8 mice). Arrow indicates scattered GFP+EMT tumor cells in the primary tumor. e , Q-RT-PCR analysis of relative expression of EMT markers in RFP+ and GFP+ cells sorted from orthotopic Tri-PyMT primary tumors. GAPDH served as the internal control. Data are reported as mean ± SEM, n=4 primary tumors.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: The EMT lineage tracing system reports EMT in tumor cells with high fidelity a , Scatter plots from flow cytometry analysis of Tri-PyMT primary tumor cells, depicting GFP+ and RFP+ populations in the primary tumor immediately after sorting of RFP+ cells (P1), and after 10 passages in culture with 10% FBS (P10+10%FBS). Numbers indicate the percentage of RFP+ and GFP+ cells in the total population. b , Phase contrast/fluorescent overlay image of Tri-PyMT cells in culture. c , Western blot of sorted RFP+ and GFP+ Tri-PyMT cells for E-cadherin, vimentin and β-actin as a loading control. Representative of 2 individual experiments. For gel source data, see Supplementary Figure 1 . d , Representative imaging of GFP+ and RFP+ tumor cells in primary tumors (PT) and lung metastases (LM) in the orthotopic model (n=8 mice). Arrow indicates scattered GFP+EMT tumor cells in the primary tumor. e , Q-RT-PCR analysis of relative expression of EMT markers in RFP+ and GFP+ cells sorted from orthotopic Tri-PyMT primary tumors. GAPDH served as the internal control. Data are reported as mean ± SEM, n=4 primary tumors.

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry, Western Blot, Imaging, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

    Establishing an orthotopic model with sorted RFP+ Tri-PyMT cells a , Flow cytometry plots show Tri-PyMT cells before and after sorting for RFP+ cells. Numbers indicate the percentage and purity of RFP+ cells used for establishing orthotopic breast tumors in mice. b , Schematic of the orthotopic breast tumor model with sorted RFP+ Tri-PyMT cells. Cells are injected into the mammary gland of wild type mice to generate primary breast tumors, resection of primary tumor at 4 weeks and lung metastases evaluation in another 4 weeks. c , Characterization of tumor cells in the primary tumor, disseminated tumor cells (DTCs) and tumor cells in the lung metastasis of the Tri-PyMT orthotopic model. Sections of primary tumors and lungs from Tri-PyMT orthotopic mice were immunostained for E-cadherin and Vimentin (in white pseudocolor). Essentially all RFP+ tumor cells are detected as E-cad+/Vim−, while the scattered GFP+ tumor cells in the primary tumor are E-Cad−/Vim+ (as indicated by arrows in the top panel). Representative images are shown (n=8). d, Plot shows the percentage of GFP+ cells out of total tumor cells (GFP+ plus RFP+, n=6).

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Establishing an orthotopic model with sorted RFP+ Tri-PyMT cells a , Flow cytometry plots show Tri-PyMT cells before and after sorting for RFP+ cells. Numbers indicate the percentage and purity of RFP+ cells used for establishing orthotopic breast tumors in mice. b , Schematic of the orthotopic breast tumor model with sorted RFP+ Tri-PyMT cells. Cells are injected into the mammary gland of wild type mice to generate primary breast tumors, resection of primary tumor at 4 weeks and lung metastases evaluation in another 4 weeks. c , Characterization of tumor cells in the primary tumor, disseminated tumor cells (DTCs) and tumor cells in the lung metastasis of the Tri-PyMT orthotopic model. Sections of primary tumors and lungs from Tri-PyMT orthotopic mice were immunostained for E-cadherin and Vimentin (in white pseudocolor). Essentially all RFP+ tumor cells are detected as E-cad+/Vim−, while the scattered GFP+ tumor cells in the primary tumor are E-Cad−/Vim+ (as indicated by arrows in the top panel). Representative images are shown (n=8). d, Plot shows the percentage of GFP+ cells out of total tumor cells (GFP+ plus RFP+, n=6).

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Injection

    Characterization of the primary tumor and lung metastasis of Tri-PyMT mice Sections of primary tumors (a) and lungs (b) from Tri-PyMT mice were immunostained for E-cadherin (E-cad, top), vimentin (Vim, middle) and CD45 (bottom) in white psuedocolor. Representative images are shown (n > 5 mice). Note the co-localization of PyMT with RFP, and CD45 with GFP (as indicated by arrows) in both primary tumors and lung metastases.

    Journal: Nature

    Article Title: EMT is not required for lung metastasis but contributes to chemoresistance

    doi: 10.1038/nature15748

    Figure Lengend Snippet: Characterization of the primary tumor and lung metastasis of Tri-PyMT mice Sections of primary tumors (a) and lungs (b) from Tri-PyMT mice were immunostained for E-cadherin (E-cad, top), vimentin (Vim, middle) and CD45 (bottom) in white psuedocolor. Representative images are shown (n > 5 mice). Note the co-localization of PyMT with RFP, and CD45 with GFP (as indicated by arrows) in both primary tumors and lung metastases.

    Article Snippet: Western blotting was performed using antibodies specific for E-cadherin (clone DECMA-1), vimentin (clone RV202, BD Pharmingen), and β-actin (clone AC-15, Sigma-Aldrich).

    Techniques: Mouse Assay

    Immunocytochemical staining of GS-1 cells using antibodies against (a) cytokeratin, (b) vimentin, (c) fibronectin, and (d) desmin (green). The cell nuclei were counterstained with diaminophenylindole (DAPI; blue) (bar=100 µ m).

    Journal: The Journal of Veterinary Medical Science

    Article Title: Assessment of fish iridoviruses using a novel cell line GS-1, derived from the spleen of orange-spotted grouper Epinephelus coioides (Hamilton) and susceptible to ranavirus and megalocytivirus

    doi: 10.1292/jvms.18-0078

    Figure Lengend Snippet: Immunocytochemical staining of GS-1 cells using antibodies against (a) cytokeratin, (b) vimentin, (c) fibronectin, and (d) desmin (green). The cell nuclei were counterstained with diaminophenylindole (DAPI; blue) (bar=100 µ m).

    Article Snippet: Briefly, the cells were grown on coverslips in six-well plates for 24 hr and then fixed with 4% paraformaldehyde at 4°C for 1 hr and permeabilized with 0.2% Triton-X 100 at room temperature for 15 min. After blocking with 2% bovine serum albumin (BSA) in PBS at room temperature for 2 hr, the cells were then incubated with four different primary antibodies of mouse-anti-cytokeratin, mouse-anti-vimentin, mouse-anti-fibronectin and rabbit-anti-desmin (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) at a dilution of 1:200 at room temperature for 2 hr to characterize the cell line.

    Techniques: Staining

    Effect of STS knockdown in T24 cells. (A) Two siRNAs for STS (si-STS A and si-STS B) reduced mRNA expression of STS in T24 cells. Relative STS expression levels are presented normalized to those in N cells. (B) Effect of siRNA knockdown on cell proliferation was evaluated using T24 cells. Each cell count was normalized to that of the N group. No significant difference was observed between si-STS-transfected and N cells. Effect of siRNA knockdown on cell migration and invasion ability was also evaluated using T24 cells. si-STS A- and B-transfected cells exhibited significantly reduced (C) migration and (D) invasion abilities compared with N cells. Scale bar indicates 500 µm. (E and F) Reverse transcription-quantitative polymerase chain reaction analyses of T24 cells transfected with siRNAs. Each gene expression level was normalized to that in N cells. (E) E-cadherin was upregulated and (F) vimentin was significantly downregulated by STS knockdown. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Steroid sulfatase promotes invasion through epithelial-mesenchymal transition and predicts the progression of bladder cancer

    doi: 10.3892/etm.2018.6787

    Figure Lengend Snippet: Effect of STS knockdown in T24 cells. (A) Two siRNAs for STS (si-STS A and si-STS B) reduced mRNA expression of STS in T24 cells. Relative STS expression levels are presented normalized to those in N cells. (B) Effect of siRNA knockdown on cell proliferation was evaluated using T24 cells. Each cell count was normalized to that of the N group. No significant difference was observed between si-STS-transfected and N cells. Effect of siRNA knockdown on cell migration and invasion ability was also evaluated using T24 cells. si-STS A- and B-transfected cells exhibited significantly reduced (C) migration and (D) invasion abilities compared with N cells. Scale bar indicates 500 µm. (E and F) Reverse transcription-quantitative polymerase chain reaction analyses of T24 cells transfected with siRNAs. Each gene expression level was normalized to that in N cells. (E) E-cadherin was upregulated and (F) vimentin was significantly downregulated by STS knockdown. *P

    Article Snippet: The catalogue numbers for the primers used for qPCR were as follows: STS, Hs00996676_m1; E-cadherin, Hs01023894_m1; Vimentin, Hs00185584_m1; GAPDH, Hs00266705_g1 (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Cell Counting, Transfection, Migration, Real-time Polymerase Chain Reaction

    Prolonged respiratory HDM exposure induces epithelial-to-mesenchymal transition. Lung sections (15 µm thick) were prepared from control mice and mice exposed to HDM for 5, 10 or 15 weeks and immunofluorescent staining for the co-expression of the LacZ reporter in airway epithelial cells with α-SMA and occludin (A–D) and with vimentin and E-cadherin (E–H) was performed. Scale bars 10 µm. Quantification of lung fibrosis was performed by morphometric analysis of lung sections stained for α-SMA (I) or LacZ and vimentin (J). * p

    Journal: PLoS ONE

    Article Title: Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

    doi: 10.1371/journal.pone.0016175

    Figure Lengend Snippet: Prolonged respiratory HDM exposure induces epithelial-to-mesenchymal transition. Lung sections (15 µm thick) were prepared from control mice and mice exposed to HDM for 5, 10 or 15 weeks and immunofluorescent staining for the co-expression of the LacZ reporter in airway epithelial cells with α-SMA and occludin (A–D) and with vimentin and E-cadherin (E–H) was performed. Scale bars 10 µm. Quantification of lung fibrosis was performed by morphometric analysis of lung sections stained for α-SMA (I) or LacZ and vimentin (J). * p

    Article Snippet: The primary antibodies used in this analysis were: rabbit anti-CAR (1∶500), mouse anti-E-cadherin (1∶500; clone 36, BD Biosciences), mouse anti-vimentin (1∶50, 3B4, DAKO), rat anti-occludin (1∶3, clone MOC37), mouse anti-SNAIL1 (1∶3) and rabbit anti-phosphorylated Smad3 (1∶100; #9520, Cell Signaling Technology, Danvers, MA).

    Techniques: Mouse Assay, Staining, Expressing

    Induction of EMT in A549 cells. Progression through EMT was evaluated in the adenocarcinoma-derived human lung epithelial cell line A549 72h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J) ), α-SMA (K) and Snail1 (L) was assessed by qPCR.* p

    Journal: PLoS ONE

    Article Title: Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

    doi: 10.1371/journal.pone.0016175

    Figure Lengend Snippet: Induction of EMT in A549 cells. Progression through EMT was evaluated in the adenocarcinoma-derived human lung epithelial cell line A549 72h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J) ), α-SMA (K) and Snail1 (L) was assessed by qPCR.* p

    Article Snippet: The primary antibodies used in this analysis were: rabbit anti-CAR (1∶500), mouse anti-E-cadherin (1∶500; clone 36, BD Biosciences), mouse anti-vimentin (1∶50, 3B4, DAKO), rat anti-occludin (1∶3, clone MOC37), mouse anti-SNAIL1 (1∶3) and rabbit anti-phosphorylated Smad3 (1∶100; #9520, Cell Signaling Technology, Danvers, MA).

    Techniques: Derivative Assay, Marker, Staining, Expressing, Real-time Polymerase Chain Reaction

    Partial induction of EMT in 16HBE14o- cells. (A–I) Progression through EMT was evaluated in the phenotypically normal human lung epithelial cell line 16HBE14o- 72 h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J), α-SMA (K) and Snail1 (L) was assessed by qPCR. * p

    Journal: PLoS ONE

    Article Title: Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

    doi: 10.1371/journal.pone.0016175

    Figure Lengend Snippet: Partial induction of EMT in 16HBE14o- cells. (A–I) Progression through EMT was evaluated in the phenotypically normal human lung epithelial cell line 16HBE14o- 72 h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J), α-SMA (K) and Snail1 (L) was assessed by qPCR. * p

    Article Snippet: The primary antibodies used in this analysis were: rabbit anti-CAR (1∶500), mouse anti-E-cadherin (1∶500; clone 36, BD Biosciences), mouse anti-vimentin (1∶50, 3B4, DAKO), rat anti-occludin (1∶3, clone MOC37), mouse anti-SNAIL1 (1∶3) and rabbit anti-phosphorylated Smad3 (1∶100; #9520, Cell Signaling Technology, Danvers, MA).

    Techniques: Marker, Staining, Expressing, Real-time Polymerase Chain Reaction

    Models of epithelial and mesenchymal cells. Images of vehicle (10 mM citric acid)-treated and TGFβ1-treated (20 ng/ml for 4 days) A549 cells ( a ), MCF-7 cells ( c ) and BEAS-2B ( e ) cells. Images of A549 ( b ) and MCF-7 ( d ) cultured in the presence of 10% or 1% FBS for 4 days. Images of serum-supplemented (+10% FBS) and serum-starved (-FBS) (bottom) BEAS-2B cells ( f ) after 7 days of serum starvation. Western blot analysis of β-actin, GAPDH, E-cadherin, N-cadherin and Vimentin in the three cell model cell lines ( d–f ). N-cadherin and Vimentin were not detectable in MCF-7 and BEAS-2B cells.

    Journal: Scientific Reports

    Article Title: A novel mechanism of plasminogen activation in epithelial and mesenchymal cells

    doi: 10.1038/s41598-018-32433-y

    Figure Lengend Snippet: Models of epithelial and mesenchymal cells. Images of vehicle (10 mM citric acid)-treated and TGFβ1-treated (20 ng/ml for 4 days) A549 cells ( a ), MCF-7 cells ( c ) and BEAS-2B ( e ) cells. Images of A549 ( b ) and MCF-7 ( d ) cultured in the presence of 10% or 1% FBS for 4 days. Images of serum-supplemented (+10% FBS) and serum-starved (-FBS) (bottom) BEAS-2B cells ( f ) after 7 days of serum starvation. Western blot analysis of β-actin, GAPDH, E-cadherin, N-cadherin and Vimentin in the three cell model cell lines ( d–f ). N-cadherin and Vimentin were not detectable in MCF-7 and BEAS-2B cells.

    Article Snippet: Vimentin (Sigma-Aldrich goat polyclonal anti-Vimentin, V4630, 1:1000) 58 kDa.

    Techniques: Cell Culture, Western Blot

    Analysis of vimentin distribution in analyzed cells. A) Vimentin was detected by anti-vimentin at the cell surface of HT22 cells transfected with siRNA for 48 h at 37°C by FACS analysis. B) Confocal microscopy of vimentin in HT22 cells transfected for 48 h at 37°C. The green (oregon green 488) anti-vimentin, DNA staining in blue (Dapi), rhodamine red-staining for actin and a merge image are shown for each panel. In the enlarged images the cell boundaries are shown. Significant difference between the vimentin distribution was detected for the transfected cells (lower panel) in comparison to the control (upper panel). Scale bar = 20 µM. C) Detection of vimentin at the cell surface of J774A.1 cells transfected with siRNA. D) Confocal microscopy of vimentin distribution in J774A.1 cells transfected for 48 h.

    Journal: PLoS ONE

    Article Title: Vimentin Mediates Uptake of C3 Exoenzyme

    doi: 10.1371/journal.pone.0101071

    Figure Lengend Snippet: Analysis of vimentin distribution in analyzed cells. A) Vimentin was detected by anti-vimentin at the cell surface of HT22 cells transfected with siRNA for 48 h at 37°C by FACS analysis. B) Confocal microscopy of vimentin in HT22 cells transfected for 48 h at 37°C. The green (oregon green 488) anti-vimentin, DNA staining in blue (Dapi), rhodamine red-staining for actin and a merge image are shown for each panel. In the enlarged images the cell boundaries are shown. Significant difference between the vimentin distribution was detected for the transfected cells (lower panel) in comparison to the control (upper panel). Scale bar = 20 µM. C) Detection of vimentin at the cell surface of J774A.1 cells transfected with siRNA. D) Confocal microscopy of vimentin distribution in J774A.1 cells transfected for 48 h.

    Article Snippet: Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA).

    Techniques: Transfection, FACS, Confocal Microscopy, Staining

    Binding of C3 to the rod domain of vimentin. His-tagged vimentin domains (A) head: amino acids 1 to 101; (B) rod: amino acids 102 to 410; (C) tail: amino acids 411 to 466 were expressed in E. coli . Crude extracts from non-induced E. coli (lane 1), crude extracts from induced E. coli (lane 2) and His-tagged vimentin domains purified through Ni-IDA column (lane 3) were separated by 15% SDS-PAGE and electroblotted. Left panels of A, B and C show probing with penta-His polyclonal antibody to detect vimentin domains. Right panels of A, B and C show probing with anti-C3 to detect bound C3. Predicted apparent mol masses of each vimentin domain are indicated by arrow. Arrows indicate the protein of interest. D) Immunoprecipitation of C3 with C3 antibody. Full length recombinant vimentin was incubated with C3 in solution. The vimentin-C3 complex was immunoprecipitated with C3 antibody. P = pellet of agarose beads, S = supernatant. E) Immunoprecipitation of vimentin with C3 antibody and detection of vimentin. P = pellet of agarose beads, S = supernatant. F) For imaging vimentin and C3 at the cell surface (upper panel), cells were incubated with C3-FITC for 1 h at 4°C. After washing with PBS cells were fixed (without permeabilization) and incubated with vimentin antibody following by an Alexa-555 conjugated secondary antibody. For imaging intracellular vimentin (lower panel), cells were fixed, permeabilized and incubated as described above. For imaging intracellular C3, living cells were incubated with C3-FITC for 1 h at 37°C. After washing with PBS cells were fixed, permeabilized with 0.02% saponin for 30 min and imaged by confocal microscopy.

    Journal: PLoS ONE

    Article Title: Vimentin Mediates Uptake of C3 Exoenzyme

    doi: 10.1371/journal.pone.0101071

    Figure Lengend Snippet: Binding of C3 to the rod domain of vimentin. His-tagged vimentin domains (A) head: amino acids 1 to 101; (B) rod: amino acids 102 to 410; (C) tail: amino acids 411 to 466 were expressed in E. coli . Crude extracts from non-induced E. coli (lane 1), crude extracts from induced E. coli (lane 2) and His-tagged vimentin domains purified through Ni-IDA column (lane 3) were separated by 15% SDS-PAGE and electroblotted. Left panels of A, B and C show probing with penta-His polyclonal antibody to detect vimentin domains. Right panels of A, B and C show probing with anti-C3 to detect bound C3. Predicted apparent mol masses of each vimentin domain are indicated by arrow. Arrows indicate the protein of interest. D) Immunoprecipitation of C3 with C3 antibody. Full length recombinant vimentin was incubated with C3 in solution. The vimentin-C3 complex was immunoprecipitated with C3 antibody. P = pellet of agarose beads, S = supernatant. E) Immunoprecipitation of vimentin with C3 antibody and detection of vimentin. P = pellet of agarose beads, S = supernatant. F) For imaging vimentin and C3 at the cell surface (upper panel), cells were incubated with C3-FITC for 1 h at 4°C. After washing with PBS cells were fixed (without permeabilization) and incubated with vimentin antibody following by an Alexa-555 conjugated secondary antibody. For imaging intracellular vimentin (lower panel), cells were fixed, permeabilized and incubated as described above. For imaging intracellular C3, living cells were incubated with C3-FITC for 1 h at 37°C. After washing with PBS cells were fixed, permeabilized with 0.02% saponin for 30 min and imaged by confocal microscopy.

    Article Snippet: Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA).

    Techniques: Binding Assay, Purification, SDS Page, Immunoprecipitation, Recombinant, Incubation, Imaging, Confocal Microscopy

    Vimentin is present at the cell surface of HT22 cells and J774A.1 macrophages. A) Intact HT22 cells were biotinylated for 1 h at 4°C. Whole cell lysates, cytosolic and particulate fractions were prepared. In addition, cell surface biotinylated proteins were enriched by precipitation with NeutrAvidin beads. The fractions and precipitation, respectively, were immunoblotted and probed with anti-vimentin. Biotinylation fraction represents the extracellular proteins exclusively. One representative Western blot experiment is shown (n = 3 independent experiments). Presence of vimentin at the cell surface of HT22 cells (B) and J774A.1 cells (C) was analyzed by FACS cytometry using anti-vimentin. Oregon green-488 conjugated goat anti-rabbit antibody alone served as negative control. Untreated cells were used as control.

    Journal: PLoS ONE

    Article Title: Vimentin Mediates Uptake of C3 Exoenzyme

    doi: 10.1371/journal.pone.0101071

    Figure Lengend Snippet: Vimentin is present at the cell surface of HT22 cells and J774A.1 macrophages. A) Intact HT22 cells were biotinylated for 1 h at 4°C. Whole cell lysates, cytosolic and particulate fractions were prepared. In addition, cell surface biotinylated proteins were enriched by precipitation with NeutrAvidin beads. The fractions and precipitation, respectively, were immunoblotted and probed with anti-vimentin. Biotinylation fraction represents the extracellular proteins exclusively. One representative Western blot experiment is shown (n = 3 independent experiments). Presence of vimentin at the cell surface of HT22 cells (B) and J774A.1 cells (C) was analyzed by FACS cytometry using anti-vimentin. Oregon green-488 conjugated goat anti-rabbit antibody alone served as negative control. Untreated cells were used as control.

    Article Snippet: Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA).

    Techniques: Western Blot, FACS, Cytometry, Negative Control

    Binding and uptake of C3 in cultivated cells dependent on vimentin. A) The Western blot shows the binding of C3 in presence and absence of extracellular added vimentin (n = 3 independent experiments). B) Densitometric evaluation of C3 (from A) and adjustment to the corresponding actin band are shown. C) The Western blot shows the degradation of RhoA as marker for C3 uptake and Rho-ADP-ribosylation. HT22 cells were treated with C3 (500 nM) alone or C3 (500 nM) plus vimentin (1 ng/µl) for 48 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. One representative Western blot experiment is shown (n = 3 independent experiments). D) Densitometric evaluation of RhoA (from C) and adjustment to the corresponding actin band are shown; the bars give the relative RhoA level. E) HT22 cells were incubated with C3 (500 nM) or C3 (500 nM) plus 1 ng/µl of either vimentin head-, rod- or tail-domain for 48 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. Decreased signal of RhoA reflects degradation of RhoA after ADP-ribosylation and thus, enhanced C3 uptake. One representative Western blot experiment is shown (n = 3 independent experiments). F) Densitometric evaluation of RhoA (from E) and adjustment to the corresponding actin band are shown; the bars give the relative RhoA level. G) J774A.1 macrophages were treated with C3 (500 nM) alone or C3 (500 nM) plus vimentin (1 ng/µl) for 2 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. One representative Western blot experiment is shown (n = 3 independent experiments). H) Primary astrocytes were exposed to C3 (300 nM) alone or a combination of C3 (300 nM) with different concentrations of vimentin (0.2, 2 and 20 ng/µl) for 6 h at 37°C. After incubation time the astrocytes were stained for the intermediate filament protein GFAP to visualize morphological changes.

    Journal: PLoS ONE

    Article Title: Vimentin Mediates Uptake of C3 Exoenzyme

    doi: 10.1371/journal.pone.0101071

    Figure Lengend Snippet: Binding and uptake of C3 in cultivated cells dependent on vimentin. A) The Western blot shows the binding of C3 in presence and absence of extracellular added vimentin (n = 3 independent experiments). B) Densitometric evaluation of C3 (from A) and adjustment to the corresponding actin band are shown. C) The Western blot shows the degradation of RhoA as marker for C3 uptake and Rho-ADP-ribosylation. HT22 cells were treated with C3 (500 nM) alone or C3 (500 nM) plus vimentin (1 ng/µl) for 48 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. One representative Western blot experiment is shown (n = 3 independent experiments). D) Densitometric evaluation of RhoA (from C) and adjustment to the corresponding actin band are shown; the bars give the relative RhoA level. E) HT22 cells were incubated with C3 (500 nM) or C3 (500 nM) plus 1 ng/µl of either vimentin head-, rod- or tail-domain for 48 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. Decreased signal of RhoA reflects degradation of RhoA after ADP-ribosylation and thus, enhanced C3 uptake. One representative Western blot experiment is shown (n = 3 independent experiments). F) Densitometric evaluation of RhoA (from E) and adjustment to the corresponding actin band are shown; the bars give the relative RhoA level. G) J774A.1 macrophages were treated with C3 (500 nM) alone or C3 (500 nM) plus vimentin (1 ng/µl) for 2 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. One representative Western blot experiment is shown (n = 3 independent experiments). H) Primary astrocytes were exposed to C3 (300 nM) alone or a combination of C3 (300 nM) with different concentrations of vimentin (0.2, 2 and 20 ng/µl) for 6 h at 37°C. After incubation time the astrocytes were stained for the intermediate filament protein GFAP to visualize morphological changes.

    Article Snippet: Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA).

    Techniques: Binding Assay, Western Blot, Marker, Incubation, Staining

    Uptake of C3 in HT22 and J744A.1 cells is dependent on vimentin distribution and integrity. A) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into HT22 cells detected as RhoA degradation (induced by C3-catalysed ADP-ribosylation). In a pulse-chase experiment, HT22 cells were incubated with C3 (500 nM) at 4°C for 60 min. Afterwards unbound C3 was removed by washing the cells three times with PBS and fresh medium was added. Cells were then cultivated for further 48 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). B) Cellular levels of RhoA proteins were quantified by densitometric evaluation of RhoA (from A) and adjusted to the corresponding actin band. C) HT22 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 24 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin. C3 alone causes a complete mol weight shift of RhoA in SDS-PAGE. Western blot analysis of one representative experiment is shown (n = 3 independent experiments). D) RhoA shift (indicative of Rho-ADP-ribosylation) by quantified by densitometric evaluation of RhoA (from C) and adjusted to the corresponding β-actin signal. E) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into J774A.1 cells detected as incomplete RhoA ADP-ribosylation. J774A.1 macrophages were incubated with C3 (500 nM) at 37°C for 4 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). F) J774A.1 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 4 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin.

    Journal: PLoS ONE

    Article Title: Vimentin Mediates Uptake of C3 Exoenzyme

    doi: 10.1371/journal.pone.0101071

    Figure Lengend Snippet: Uptake of C3 in HT22 and J744A.1 cells is dependent on vimentin distribution and integrity. A) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into HT22 cells detected as RhoA degradation (induced by C3-catalysed ADP-ribosylation). In a pulse-chase experiment, HT22 cells were incubated with C3 (500 nM) at 4°C for 60 min. Afterwards unbound C3 was removed by washing the cells three times with PBS and fresh medium was added. Cells were then cultivated for further 48 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). B) Cellular levels of RhoA proteins were quantified by densitometric evaluation of RhoA (from A) and adjusted to the corresponding actin band. C) HT22 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 24 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin. C3 alone causes a complete mol weight shift of RhoA in SDS-PAGE. Western blot analysis of one representative experiment is shown (n = 3 independent experiments). D) RhoA shift (indicative of Rho-ADP-ribosylation) by quantified by densitometric evaluation of RhoA (from C) and adjusted to the corresponding β-actin signal. E) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into J774A.1 cells detected as incomplete RhoA ADP-ribosylation. J774A.1 macrophages were incubated with C3 (500 nM) at 37°C for 4 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). F) J774A.1 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 4 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin.

    Article Snippet: Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA).

    Techniques: Pulse Chase, Incubation, Generated, SDS Page, Western Blot

    Knock down of vimentin in hippocampal HT22 cells and J774A.1 macrophages. A) HT22 cells were transfected with siRNA for 48 h (scr = scrambled, Vim = vimentin). Vimentin and β-actin were detected by Western blot analysis of cell lysates. B) After siRNA transfection for 48 h, HT22 cells were exposed to C3 (100 nM) for 1 h at 4°C. Bound C3 was detected in Western blot with anti-C3. β-actin was used as internal control. C) Densitometric evaluation of bound C3 (from B) and adjustment to the corresponding actin band are shown; the bars give the relative C3 binding. D) HT22 cells transfected with siRNA for 48 h were incubated with C3-E174Q-FITC (500 nM) for 1 h at 4°C and bound C3-E174Q-FITC was analyzed by FACS cytometry. E – G) Same experiments for J774A.1 macrophages. E) Knock down of vimentin. F) Binding of C3 to cells with vimentin knock down. G) Densitometric evaluation of F. H) Binding of C3-E174Q-FITC to cells with vimentin knock down and FACS analysis.

    Journal: PLoS ONE

    Article Title: Vimentin Mediates Uptake of C3 Exoenzyme

    doi: 10.1371/journal.pone.0101071

    Figure Lengend Snippet: Knock down of vimentin in hippocampal HT22 cells and J774A.1 macrophages. A) HT22 cells were transfected with siRNA for 48 h (scr = scrambled, Vim = vimentin). Vimentin and β-actin were detected by Western blot analysis of cell lysates. B) After siRNA transfection for 48 h, HT22 cells were exposed to C3 (100 nM) for 1 h at 4°C. Bound C3 was detected in Western blot with anti-C3. β-actin was used as internal control. C) Densitometric evaluation of bound C3 (from B) and adjustment to the corresponding actin band are shown; the bars give the relative C3 binding. D) HT22 cells transfected with siRNA for 48 h were incubated with C3-E174Q-FITC (500 nM) for 1 h at 4°C and bound C3-E174Q-FITC was analyzed by FACS cytometry. E – G) Same experiments for J774A.1 macrophages. E) Knock down of vimentin. F) Binding of C3 to cells with vimentin knock down. G) Densitometric evaluation of F. H) Binding of C3-E174Q-FITC to cells with vimentin knock down and FACS analysis.

    Article Snippet: Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA).

    Techniques: Transfection, Western Blot, Binding Assay, Incubation, FACS, Cytometry