Structured Review

Thermo Fisher fascin
Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of <t>PKC-ε</t> and PDGF-Rα and miR-145 repression of <t>fascin,</t> allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.
Fascin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
fascin - by Bioz Stars, 2020-07
93/100 stars

Images

1) Product Images from "MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro"

Article Title: MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200912096

Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of PKC-ε and PDGF-Rα and miR-145 repression of fascin, allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.
Figure Legend Snippet: Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of PKC-ε and PDGF-Rα and miR-145 repression of fascin, allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.

Techniques Used: Migration, Expressing, Activity Assay

miR-143 and -145 targets. (A) Representative immunoblot of primary VSMCs isolated from miR-143(145) KO mice transduced with adenovirus (Ad-miR) with an empty expression cassette (−) or expressing miR-143, -145, or -208. (B) Luciferase reporter assay on 3T3 cells performed by cotransfection of 20 nM miR-143, miR-145, or scrambled oligonucleotide (+) with a renilla reporter gene linked to 10 ng WT (wt) or mutated (mt) 3′ UTR of PKC-ε, PDGF-Rα, or fascin. All measurements were calculated as the percentage of control (WT 3′ UTR), and error bars were calculated as propagated standard errors of the mean of triplicate measurements from each experiment. *, P
Figure Legend Snippet: miR-143 and -145 targets. (A) Representative immunoblot of primary VSMCs isolated from miR-143(145) KO mice transduced with adenovirus (Ad-miR) with an empty expression cassette (−) or expressing miR-143, -145, or -208. (B) Luciferase reporter assay on 3T3 cells performed by cotransfection of 20 nM miR-143, miR-145, or scrambled oligonucleotide (+) with a renilla reporter gene linked to 10 ng WT (wt) or mutated (mt) 3′ UTR of PKC-ε, PDGF-Rα, or fascin. All measurements were calculated as the percentage of control (WT 3′ UTR), and error bars were calculated as propagated standard errors of the mean of triplicate measurements from each experiment. *, P

Techniques Used: Isolation, Mouse Assay, Transduction, Expressing, Luciferase, Reporter Assay, Cotransfection

2) Product Images from "Cellular and Tissue Architecture of Conjunctival Membranes in Epidemic Keratoconjunctivitis"

Article Title: Cellular and Tissue Architecture of Conjunctival Membranes in Epidemic Keratoconjunctivitis

Journal: Ocular immunology and inflammation

doi: 10.3109/09273948.2010.498658

Leukocyte phenotype of conjunctival membranes in EKC. Representative photomicrographs of ( A ) CD68, ( B ) myeloperoxidase, ( C ) CD4, ( D ) CD8, ( E ) CD20, and ( F ) fascin. Scale bar, 50 μM.
Figure Legend Snippet: Leukocyte phenotype of conjunctival membranes in EKC. Representative photomicrographs of ( A ) CD68, ( B ) myeloperoxidase, ( C ) CD4, ( D ) CD8, ( E ) CD20, and ( F ) fascin. Scale bar, 50 μM.

Techniques Used:

Related Articles

Staining:

Article Title: Association of Fascin and matrix metalloproteinase-9 expression with poor prognostic parameters in breast carcinoma of Egyptian women
Article Snippet: .. Immunohistochemical staining was performed using primary antibodies; mouse monoclonal anti-fascin (Clone: FCN01; 1:200 dilution; Thermo Fisher Scientific Inc., Fremont, CA) and mouse monoclonal anti MMP-9 (Clone: GE-213;1:200 dilution; Thermo Fisher Scientific Inc., Fremont, CA). .. Avidin-Biotin immunoperoxidase complex technique was used according to Hsu et al. [ ] by applying the super sensitive detection kit (Biogenex, CA, USA).

Immunohistochemistry:

Article Title: Association of Fascin and matrix metalloproteinase-9 expression with poor prognostic parameters in breast carcinoma of Egyptian women
Article Snippet: .. Immunohistochemical staining was performed using primary antibodies; mouse monoclonal anti-fascin (Clone: FCN01; 1:200 dilution; Thermo Fisher Scientific Inc., Fremont, CA) and mouse monoclonal anti MMP-9 (Clone: GE-213;1:200 dilution; Thermo Fisher Scientific Inc., Fremont, CA). .. Avidin-Biotin immunoperoxidase complex technique was used according to Hsu et al. [ ] by applying the super sensitive detection kit (Biogenex, CA, USA).

other:

Article Title: Cellular and Tissue Architecture of Conjunctival Membranes in Epidemic Keratoconjunctivitis
Article Snippet: Antibodies against CD4, myeloperoxidase, vascular endothelial growth factor (VEGF), fascin, and transforming growth factor-β (TGF-β) were purchased from Lab Vision Corporation (Fremont, CA).

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  • 91
    Thermo Fisher fascin
    Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of <t>PKC-ε</t> and PDGF-Rα and miR-145 repression of <t>fascin,</t> allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.
    Fascin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fascin/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fascin - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    90
    Thermo Fisher fascin 1
    Polarization of actin and DC-specific actin bundling protein. <t>Fascin-1</t> is associated with T reg cell–mediated suppression (A) Maximum-intensity projection (MIP) SIM images of a T reg cell or an OT-II T cell coupled with a DC2.4 cell pulsed with OVA, stained for DNA with DAPI, anti-α-tubulin and phalloidin. White arrows highlight the increased thickening of F-actin at the T–DC interface. Hand-traced boundaries of T cells according to DIC are shown to the right here, as well as in the subsequent images. Bars, 5 µm. Representative of at least 10 independent images from four independent stainings. (right) SiR-actin/DAPI-labeled DCs in mixture with T cells (DAPI only) under conventional microscope. (B, top) MIP SIM images of conjugates between T reg cells and DC2.4 cells transfected with control or Fascin-1 siRNA. Conjugates were stained with DAPI, phalloidin, and anti–Fascin-1 antibody. Red boxes covering the T–DC contact sites were used for statistical analyses of Facsin-1 intensity. (bottom) Fascin-1 intensities at T reg cell–DC and OT-II–DC contact sites, as indicated by red boxes in the left. (bottom right) Ratios of mean Fascin-1 intensities in and out of the junction areas. Representative of at least 10 independent images from four independent stainings. (C) MIP SIM images and representative optical sections (stack 1–3) of conjugates between T reg cell (top two rows) or OT-II (bottom two rows) cells and DC2.4 cells. Arrows highlight areas of intermingled Fascin-1 and F-actin bundling, magnified in the inserts. Representative of at least 10 independent images from three independent stainings. (D) As in B, Fascin-1 distribution in three T reg cell–BMDC or OT-II-BMDC pairs was visualized by SIM (top) and quantitatively analyzed in the lower. Representative of three independent stainings. (E, left) Mean forces of OT-II T cells adhering to untreated, control siRNA-treated, or Fascin-1–specific siRNA-treated DC2.4 cells prepulsed with OVA. (right) Mean forces of OT-II T cells adhering to control vector-transfected or Fascin-1–overexpressing DC2.4 cells prepulsed with OVA, with or without T reg cell contact. Each is representative of at least three independent experiments. **,
    Fascin 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fascin 1/product/Thermo Fisher
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    90/100 stars
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    85
    Thermo Fisher mutant fascin 1 proteins
    Rho kinase activity promotes peripheral fascin-containing protrusions via a myosin-independent process . (A) Confocal images of C2C12 cells after 1 hour of adhesion to 50 nmol/l fibronectin (FN), either untreated or pretreated with specified inhibitors, fixed and stained for <t>fascin-1.</t> Arrowheads indicate examples of peripheral fascin-actin bundles in Y27632-treated cells. Scale bars, 10 μm. (B) Confocal images of C2C12 cells transiently expressing green fluorescent protein (GFP)-caldesmon or an inactive GFP-caldesmon-445 mutant after 1 hour of adhesion to 50 nmol/l FN. Cells were fixed and stained either for F-actin (left panels) or fascin-1 (right panels). In the anti-fascin-1 stained samples, arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Quantification of peripheral fascin-1 bundles/cell under the conditions shown in (A) and (B ). Data are from 75 to 125 cells/condition and 3 independent experiments. * P
    Mutant Fascin 1 Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of PKC-ε and PDGF-Rα and miR-145 repression of fascin, allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.

    Journal: The Journal of Cell Biology

    Article Title: MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

    doi: 10.1083/jcb.200912096

    Figure Lengend Snippet: Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of PKC-ε and PDGF-Rα and miR-145 repression of fascin, allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.

    Article Snippet: RNA interference Smart pool siRNAs for scrambled, PKC-ε, and fascin were purchased from Thermo Fisher Scientific and transfected into Src-3T3 using Lipofectamine 2000.

    Techniques: Migration, Expressing, Activity Assay

    miR-143 and -145 targets. (A) Representative immunoblot of primary VSMCs isolated from miR-143(145) KO mice transduced with adenovirus (Ad-miR) with an empty expression cassette (−) or expressing miR-143, -145, or -208. (B) Luciferase reporter assay on 3T3 cells performed by cotransfection of 20 nM miR-143, miR-145, or scrambled oligonucleotide (+) with a renilla reporter gene linked to 10 ng WT (wt) or mutated (mt) 3′ UTR of PKC-ε, PDGF-Rα, or fascin. All measurements were calculated as the percentage of control (WT 3′ UTR), and error bars were calculated as propagated standard errors of the mean of triplicate measurements from each experiment. *, P

    Journal: The Journal of Cell Biology

    Article Title: MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

    doi: 10.1083/jcb.200912096

    Figure Lengend Snippet: miR-143 and -145 targets. (A) Representative immunoblot of primary VSMCs isolated from miR-143(145) KO mice transduced with adenovirus (Ad-miR) with an empty expression cassette (−) or expressing miR-143, -145, or -208. (B) Luciferase reporter assay on 3T3 cells performed by cotransfection of 20 nM miR-143, miR-145, or scrambled oligonucleotide (+) with a renilla reporter gene linked to 10 ng WT (wt) or mutated (mt) 3′ UTR of PKC-ε, PDGF-Rα, or fascin. All measurements were calculated as the percentage of control (WT 3′ UTR), and error bars were calculated as propagated standard errors of the mean of triplicate measurements from each experiment. *, P

    Article Snippet: RNA interference Smart pool siRNAs for scrambled, PKC-ε, and fascin were purchased from Thermo Fisher Scientific and transfected into Src-3T3 using Lipofectamine 2000.

    Techniques: Isolation, Mouse Assay, Transduction, Expressing, Luciferase, Reporter Assay, Cotransfection

    Polarization of actin and DC-specific actin bundling protein. Fascin-1 is associated with T reg cell–mediated suppression (A) Maximum-intensity projection (MIP) SIM images of a T reg cell or an OT-II T cell coupled with a DC2.4 cell pulsed with OVA, stained for DNA with DAPI, anti-α-tubulin and phalloidin. White arrows highlight the increased thickening of F-actin at the T–DC interface. Hand-traced boundaries of T cells according to DIC are shown to the right here, as well as in the subsequent images. Bars, 5 µm. Representative of at least 10 independent images from four independent stainings. (right) SiR-actin/DAPI-labeled DCs in mixture with T cells (DAPI only) under conventional microscope. (B, top) MIP SIM images of conjugates between T reg cells and DC2.4 cells transfected with control or Fascin-1 siRNA. Conjugates were stained with DAPI, phalloidin, and anti–Fascin-1 antibody. Red boxes covering the T–DC contact sites were used for statistical analyses of Facsin-1 intensity. (bottom) Fascin-1 intensities at T reg cell–DC and OT-II–DC contact sites, as indicated by red boxes in the left. (bottom right) Ratios of mean Fascin-1 intensities in and out of the junction areas. Representative of at least 10 independent images from four independent stainings. (C) MIP SIM images and representative optical sections (stack 1–3) of conjugates between T reg cell (top two rows) or OT-II (bottom two rows) cells and DC2.4 cells. Arrows highlight areas of intermingled Fascin-1 and F-actin bundling, magnified in the inserts. Representative of at least 10 independent images from three independent stainings. (D) As in B, Fascin-1 distribution in three T reg cell–BMDC or OT-II-BMDC pairs was visualized by SIM (top) and quantitatively analyzed in the lower. Representative of three independent stainings. (E, left) Mean forces of OT-II T cells adhering to untreated, control siRNA-treated, or Fascin-1–specific siRNA-treated DC2.4 cells prepulsed with OVA. (right) Mean forces of OT-II T cells adhering to control vector-transfected or Fascin-1–overexpressing DC2.4 cells prepulsed with OVA, with or without T reg cell contact. Each is representative of at least three independent experiments. **,

    Journal: The Journal of Experimental Medicine

    Article Title: Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy

    doi: 10.1084/jem.20160620

    Figure Lengend Snippet: Polarization of actin and DC-specific actin bundling protein. Fascin-1 is associated with T reg cell–mediated suppression (A) Maximum-intensity projection (MIP) SIM images of a T reg cell or an OT-II T cell coupled with a DC2.4 cell pulsed with OVA, stained for DNA with DAPI, anti-α-tubulin and phalloidin. White arrows highlight the increased thickening of F-actin at the T–DC interface. Hand-traced boundaries of T cells according to DIC are shown to the right here, as well as in the subsequent images. Bars, 5 µm. Representative of at least 10 independent images from four independent stainings. (right) SiR-actin/DAPI-labeled DCs in mixture with T cells (DAPI only) under conventional microscope. (B, top) MIP SIM images of conjugates between T reg cells and DC2.4 cells transfected with control or Fascin-1 siRNA. Conjugates were stained with DAPI, phalloidin, and anti–Fascin-1 antibody. Red boxes covering the T–DC contact sites were used for statistical analyses of Facsin-1 intensity. (bottom) Fascin-1 intensities at T reg cell–DC and OT-II–DC contact sites, as indicated by red boxes in the left. (bottom right) Ratios of mean Fascin-1 intensities in and out of the junction areas. Representative of at least 10 independent images from four independent stainings. (C) MIP SIM images and representative optical sections (stack 1–3) of conjugates between T reg cell (top two rows) or OT-II (bottom two rows) cells and DC2.4 cells. Arrows highlight areas of intermingled Fascin-1 and F-actin bundling, magnified in the inserts. Representative of at least 10 independent images from three independent stainings. (D) As in B, Fascin-1 distribution in three T reg cell–BMDC or OT-II-BMDC pairs was visualized by SIM (top) and quantitatively analyzed in the lower. Representative of three independent stainings. (E, left) Mean forces of OT-II T cells adhering to untreated, control siRNA-treated, or Fascin-1–specific siRNA-treated DC2.4 cells prepulsed with OVA. (right) Mean forces of OT-II T cells adhering to control vector-transfected or Fascin-1–overexpressing DC2.4 cells prepulsed with OVA, with or without T reg cell contact. Each is representative of at least three independent experiments. **,

    Article Snippet: SIM slices were taken every 0.1-µm apart, and data were presented as maximum intensity projection of all collected optical sections except that, for certain experiments as indicated, individual slices were selected to depict interactions between Fascin-1 and F-actin.

    Techniques: Staining, Labeling, Microscopy, Transfection, Plasmid Preparation

    Rho kinase activity promotes peripheral fascin-containing protrusions via a myosin-independent process . (A) Confocal images of C2C12 cells after 1 hour of adhesion to 50 nmol/l fibronectin (FN), either untreated or pretreated with specified inhibitors, fixed and stained for fascin-1. Arrowheads indicate examples of peripheral fascin-actin bundles in Y27632-treated cells. Scale bars, 10 μm. (B) Confocal images of C2C12 cells transiently expressing green fluorescent protein (GFP)-caldesmon or an inactive GFP-caldesmon-445 mutant after 1 hour of adhesion to 50 nmol/l FN. Cells were fixed and stained either for F-actin (left panels) or fascin-1 (right panels). In the anti-fascin-1 stained samples, arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Quantification of peripheral fascin-1 bundles/cell under the conditions shown in (A) and (B ). Data are from 75 to 125 cells/condition and 3 independent experiments. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Rho kinase activity promotes peripheral fascin-containing protrusions via a myosin-independent process . (A) Confocal images of C2C12 cells after 1 hour of adhesion to 50 nmol/l fibronectin (FN), either untreated or pretreated with specified inhibitors, fixed and stained for fascin-1. Arrowheads indicate examples of peripheral fascin-actin bundles in Y27632-treated cells. Scale bars, 10 μm. (B) Confocal images of C2C12 cells transiently expressing green fluorescent protein (GFP)-caldesmon or an inactive GFP-caldesmon-445 mutant after 1 hour of adhesion to 50 nmol/l FN. Cells were fixed and stained either for F-actin (left panels) or fascin-1 (right panels). In the anti-fascin-1 stained samples, arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Quantification of peripheral fascin-1 bundles/cell under the conditions shown in (A) and (B ). Data are from 75 to 125 cells/condition and 3 independent experiments. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Activity Assay, Staining, Expressing, Mutagenesis, Transfection

    The fascin-1/p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 interaction promotes stabilization of filopodia . (A-D) Images from confocal time-lapse movies of cells expressing mRFP-fascin-1 with (A) green fluorescent protein (GFP), (B) GFP-LIMK1 (B), (C) GFP-LIMK1T508A or (D) GFP-LIMK1D460A. see Additional files 7 to 10 (movies 4 to 7). There were 15 to 25 cells per condition analyzed in 4 independent experiments. and cells from representative movies are shown. (A-D) Boxed 15 × 15 mm regions in the lefthand panels are enlarged in the images from a series of time points in the right panels. See Additional file 3 , Figure S3, for single-channel images from (A) and (B) . Scale bars, 10 μm. (E) Kymographs of representative filopodia from cells co-expressing GFP or GFP-LIMK1 and mRFP-fascin-1. Displacement of filopodia was measured in accordance with the maximum change in position of filopodial tips over 2 minutes, as indicated by the dotted yellow lines. (F) Quantification of the maximum displacement of filopodia from cells expressing GFP, or wild-type or mutant GFP-LIMK1 and mRFP-fascin-1. Each column represents the mean from five filopodia from at least five cells per condition in four independent experiments; bars indicate SEM. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: The fascin-1/p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 interaction promotes stabilization of filopodia . (A-D) Images from confocal time-lapse movies of cells expressing mRFP-fascin-1 with (A) green fluorescent protein (GFP), (B) GFP-LIMK1 (B), (C) GFP-LIMK1T508A or (D) GFP-LIMK1D460A. see Additional files 7 to 10 (movies 4 to 7). There were 15 to 25 cells per condition analyzed in 4 independent experiments. and cells from representative movies are shown. (A-D) Boxed 15 × 15 mm regions in the lefthand panels are enlarged in the images from a series of time points in the right panels. See Additional file 3 , Figure S3, for single-channel images from (A) and (B) . Scale bars, 10 μm. (E) Kymographs of representative filopodia from cells co-expressing GFP or GFP-LIMK1 and mRFP-fascin-1. Displacement of filopodia was measured in accordance with the maximum change in position of filopodial tips over 2 minutes, as indicated by the dotted yellow lines. (F) Quantification of the maximum displacement of filopodia from cells expressing GFP, or wild-type or mutant GFP-LIMK1 and mRFP-fascin-1. Each column represents the mean from five filopodia from at least five cells per condition in four independent experiments; bars indicate SEM. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Expressing, Mutagenesis

    Activation of p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 leads to its interaction with fascin-1 and affects formation of filopodia . (A) Measurement of the interaction of wild-type or mutant forms of green fluorescent protein (GFP)-LIMK1 with monomeric red fluorescent protein (mRFP)-fascin-1 in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (B) Percentage FRET efficiency under each experimental condition. Each column represents the mean from fourteen to seventeen cells per condition and three independent experiments; bars indicate SEM. * P = 0.001 versus wildtype. (C) Role of LIMK1 activity in organization of filopodia. Live SW480 cells transiently transfected with GFP alone or GFP-LIMK1 (wild-type (WT), D460A or T508A) and mRFP-fascin-1, and protein localizations and cell edges were imaged using confocal microscopy. Arrowheads indicate points where GFP-LIMK1 and mRFP-fascin-1 colocalize in filopodia. Scale bars, 10 μm. (D) The number/cell and (E) length of filopodia were counted from images obtained as in (C) , from 12 to 20 cells per condition and 4 independent experiments. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Activation of p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 leads to its interaction with fascin-1 and affects formation of filopodia . (A) Measurement of the interaction of wild-type or mutant forms of green fluorescent protein (GFP)-LIMK1 with monomeric red fluorescent protein (mRFP)-fascin-1 in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (B) Percentage FRET efficiency under each experimental condition. Each column represents the mean from fourteen to seventeen cells per condition and three independent experiments; bars indicate SEM. * P = 0.001 versus wildtype. (C) Role of LIMK1 activity in organization of filopodia. Live SW480 cells transiently transfected with GFP alone or GFP-LIMK1 (wild-type (WT), D460A or T508A) and mRFP-fascin-1, and protein localizations and cell edges were imaged using confocal microscopy. Arrowheads indicate points where GFP-LIMK1 and mRFP-fascin-1 colocalize in filopodia. Scale bars, 10 μm. (D) The number/cell and (E) length of filopodia were counted from images obtained as in (C) , from 12 to 20 cells per condition and 4 independent experiments. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Activation Assay, Mutagenesis, Transfection, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer, Activity Assay, Confocal Microscopy

    Model for the novel pathway that regulates fascin-1/actin interaction and filopodia stability . See Discussion for details. As shown in Figure 6, active p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1/2 bound to both S39-phosphorylated and non-phosphorylated fascin-1.

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Model for the novel pathway that regulates fascin-1/actin interaction and filopodia stability . See Discussion for details. As shown in Figure 6, active p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1/2 bound to both S39-phosphorylated and non-phosphorylated fascin-1.

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques:

    Rho activity promotes the interaction of fascin-1 with actin: detection by a novel fascin-1/lifeact fluorescence resonance energy transfer (FRET) system . (A,B) Measurement of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in (A) C2C12 cells on fibronectin (FN) or (B) SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). (B) Representative images of GFP and lifetime plot in the absence of an acceptor, or in presence of mRFP-fascin-1S39D, which does not bundle F-actin. In each panel, lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) ,Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Rho activity promotes the interaction of fascin-1 with actin: detection by a novel fascin-1/lifeact fluorescence resonance energy transfer (FRET) system . (A,B) Measurement of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in (A) C2C12 cells on fibronectin (FN) or (B) SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). (B) Representative images of GFP and lifetime plot in the absence of an acceptor, or in presence of mRFP-fascin-1S39D, which does not bundle F-actin. In each panel, lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) ,Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Activity Assay, Fluorescence, Förster Resonance Energy Transfer, Transfection, Imaging, Microscopy

    Rho activity does not modulate the interaction of fascin-1 with conventional protein kinase C (cPKC) . (A) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCα- monomeric red fluorescent protein (mRFP) in C2C12 cells on fibronectin (FN). (B) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCγ-mRFP in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM.* P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Rho activity does not modulate the interaction of fascin-1 with conventional protein kinase C (cPKC) . (A) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCα- monomeric red fluorescent protein (mRFP) in C2C12 cells on fibronectin (FN). (B) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCγ-mRFP in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM.* P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Activity Assay, Transfection, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer

    Rho kinase activity promotes the interaction of fascin-1 with actin . (A) Percentage FRET efficiency of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in SW480 cells on laminin (LN) under control conditions or after inhibition of Rho kinases by Y27632. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Rho kinase activity promotes the interaction of fascin-1 with actin . (A) Percentage FRET efficiency of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in SW480 cells on laminin (LN) under control conditions or after inhibition of Rho kinases by Y27632. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Activity Assay, Inhibition

    Rho-dependent and Rho kinase-dependent interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) (A,B) . Measurement of the interaction of (A) green fluorescent protein (GFP)-LIMK1 (A), or (B) GFP-LIMK2, with monomeric red fluorescent protein (mRFP)-fascin-1S39A in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for monomeric red fluorescent protein (mRFP) (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from nine to sixteen cells per condition and three independent experiments; bars indicate SEM. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Rho-dependent and Rho kinase-dependent interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) (A,B) . Measurement of the interaction of (A) green fluorescent protein (GFP)-LIMK1 (A), or (B) GFP-LIMK2, with monomeric red fluorescent protein (mRFP)-fascin-1S39A in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for monomeric red fluorescent protein (mRFP) (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from nine to sixteen cells per condition and three independent experiments; bars indicate SEM. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Transfection, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer

    Rho inhibition modulates peripheral fascin-containing protrusions . (A) C2C12 cells (control or treated with the indicated pharmacological inhibitors), were plated onto 50 nmol/l fibronectin (FN) for 1 hour, then fixed and stained for fascin-1. Arrowheads indicate examples of fascin-containing protrusions, dotted arrow indicates fascin in association with stress fibers. Boxed areas are enlarged below. Scale bars, 10 μm. (B) Representative results of rhotekin-Rho-binding domain (RBD) pull-down of Rho-guanine triphosphate (GTP) from C2C12 cells adherent on 30 nmol/l FN or thrombospondin-1 for 1 hour, or suspended for 90 minutes over BSA-coated plastic. (C,D) Quantification of (C) numbers and (D) length of peripheral fascin bundles in C2C12 cells adherent for 1 hour on 50 nnmol/l FN after each treatment. Each column represents the mean from 70 to 100 cells from 3 independent experiments; bars indicate SEM. * P

    Journal: BMC Biology

    Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

    doi: 10.1186/1741-7007-10-72

    Figure Lengend Snippet: Rho inhibition modulates peripheral fascin-containing protrusions . (A) C2C12 cells (control or treated with the indicated pharmacological inhibitors), were plated onto 50 nmol/l fibronectin (FN) for 1 hour, then fixed and stained for fascin-1. Arrowheads indicate examples of fascin-containing protrusions, dotted arrow indicates fascin in association with stress fibers. Boxed areas are enlarged below. Scale bars, 10 μm. (B) Representative results of rhotekin-Rho-binding domain (RBD) pull-down of Rho-guanine triphosphate (GTP) from C2C12 cells adherent on 30 nmol/l FN or thrombospondin-1 for 1 hour, or suspended for 90 minutes over BSA-coated plastic. (C,D) Quantification of (C) numbers and (D) length of peripheral fascin bundles in C2C12 cells adherent for 1 hour on 50 nnmol/l FN after each treatment. Each column represents the mean from 70 to 100 cells from 3 independent experiments; bars indicate SEM. * P

    Article Snippet: For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ).

    Techniques: Inhibition, Staining, Binding Assay