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  • 91
    Agilent technologies fascin
    SIV is present in the rectal epithelium as early as four hours post infection. Serial paraffin sections of R-H4.1 (sacrificed four hours pi; A and B) and R-H16.2 (sacrificed sixteen hours pi; C and D) were labeled by IHF for gp130 (Alexa-546, A and C), <t>CD3</t> (TRITC, B and D) or <t>fascin</t> (Alexa-488, B and D) and nuclei were stained with DAPI (A–D). CR, crypt; LP, lamina propria; RL, rectal lumen. Arrows on serial sections point to intraepithelial T lymphocytes positive for SIV antigens. Cell-associated virus in the epithelial cell fraction was assayed by co-culture of cells isolated from the rectum. Results are expressed as TCID 50 per million cells. The viral load does not increase over the first four days of infection; + the TCID 50 could not be calculated due to small number of wells positive for SIV antigen (E).
    Fascin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam fascin
    cbMg show cell clearance phenotype at the single cell, protein, and functional level (a) Schematic showing single nuclei RNA-seq (Fluidigm C1). Heatmap with unsupervised clustering displays the variation in the expression levels (z-score) of 75 most differentially expressed genes across all single nuclei. (b) Bar graphs show normalized expression levels (counts per million) of selected genes for single stMg/cbMg nuclei. Experiment was repeated independently 2 times with similar results. (c) Western blot analysis of CD11b-bead-isolated microglia (75,000 cbMg/stMg from one mouse per lane). Representative blots (left, cropped to show the specific band); quantifications (right). ACTB is used as loading control. <t>FSCN1:</t> stMg: mean=1.0, SEM=0.1887; cbMg: mean=0.1792, SEM=0.08138; p=0.0018, F=5.378, t 11 =3.994. MRC1: stMg: mean=1.0, SEM=0.2132; cbMg: mean=2.051, SEM=0.16; p=0.0043, F=1.776, t 8 =3.941. AXL: stMg: mean=1.0, SEM=0.3482; cbMg: mean=11.79, SEM=2.691; p=0.0018, F=59.71, t 12 =3.977. LC3: stMg: mean=1.0, SEM=0.2328; cbMg: mean=4.647, SEM=1.179; p=0.0162, F=25.66, t 8 . (d) Schematic showing phagocytosis assay of acutely isolated adult cbMg/stMg exposed to early apoptotic cells in vitro. Quantification of the percentage of GFP+ microglia that engulfed pHrodo+ early apoptotic cells in presence/absence of phagocytosis inhibitor, Cytochalasin D (Cyt) (stMg: mean=9.055, SEM=1.761; stMg+Cyt: mean= 0.5725, SEM=0.3420; cbMg: mean=24.63, SEM=0.3789; cbMg+Cyt: mean=2.720, SEM=1.589; p brain region
    Fascin, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology fascin
    Analysis of Cdc42 and <t>fascin</t> in <t>MYC-nick–expressing</t> cells. ( A ) MYC-nick–overexpressing HFF cells show increased filopodia formation that is further enhanced when Cdc42 is activated. (Magnification: 63×.) ( B ) HCT116 cells overexpressing
    Fascin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc fascin
    Induction of <t>STAT3,</t> <t>FASCIN</t> and SURVIVIN expression by c-SRC and suppression by MLT in MCF-7 breast cancer cells. MCF-7 breast cancer cells stably transfected with the constitutively active SRC-D construct and treated with MLT (10 −9 M or 10 −8 M) for 24 h, after which the expression of t-STAT3, pSTAT3, t-FASCIN, and t-SURVIVIN levels were measured by Western blot. β-actin was used as a control for equal loading.
    Fascin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Developmental Studies Hybridoma Bank mouse anti fascin
    Model of the functions of Jumu in filopodium formation and circulating hemocyte phagocytosis. a Normal expression of Jumu maintains the levels of NimC1, Enabled and <t>Fascin</t> in a direct or indirect manner. Enabled and Fascin participate in filopodium formation. The phagocytosis receptor NimC1 is involved in the recognition of pathogens and the subcellular localization of <t>Ena.</t> b Deficiency of Jumu reduces the protein levels of NimC1, Enabled and Fascin and consequently inhibits filopodium formation and hemocyte phagocytosis. c Severe deficiency of jumu can inhibit normal hemocyte mitosis and result in enlarged multinucleated hemocytes. The loss of jumu may control the cell cycle and mitosis process by affecting the expression of the Cyclin genes png and piwi . Severe deficiency of jumu also induces the generation of lamellocytes through the activation of the Toll signaling pathway
    Mouse Anti Fascin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 88/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti fascin
    Model of the functions of Jumu in filopodium formation and circulating hemocyte phagocytosis. a Normal expression of Jumu maintains the levels of NimC1, Enabled and <t>Fascin</t> in a direct or indirect manner. Enabled and Fascin participate in filopodium formation. The phagocytosis receptor NimC1 is involved in the recognition of pathogens and the subcellular localization of <t>Ena.</t> b Deficiency of Jumu reduces the protein levels of NimC1, Enabled and Fascin and consequently inhibits filopodium formation and hemocyte phagocytosis. c Severe deficiency of jumu can inhibit normal hemocyte mitosis and result in enlarged multinucleated hemocytes. The loss of jumu may control the cell cycle and mitosis process by affecting the expression of the Cyclin genes png and piwi . Severe deficiency of jumu also induces the generation of lamellocytes through the activation of the Toll signaling pathway
    Anti Fascin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Agilent technologies fascin 1
    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
    Fascin 1, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies anti human fascin monoclonal antibody
    Immunofluorescence double-staining to phenotype the P. acnes -positive cells in non-granulomatous areas of sarcoid lungs and lymph nodes. Green signal (FITC): PAB antibody; red signals (TRITC): <t>anti-CD68</t> antibody ( a , c , e ), <t>anti-fascin</t> antibody ( b , d , f ). Results of double-staining are merged in all pictures. In the sarcoid lung, all of the P. acnes signals overlapped (yellow) with CD68-positive alveolar macrophages ( a ), whereas the fascin-positive alveolar dendritic cells did not contain small round bodies detected by PAB antibody ( b ). In the paracortical area of the sarcoid lymph node, small round body detected by PAB antibody were found in the CD68-positive macrophages ( c ), but not in the fascin-positive dendritic cells ( d ). In the sinus of the sarcoid lymph node, Hamazaki-Wesenberg bodies detected by PAB antibody overlapped with CD68-positive macrophages ( e ), but not with the fascin-positive dendritic cells ( f ). Original magnification: a – b , × 400; c – f , × 1000.
    Anti Human Fascin Monoclonal Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam monoclonal mouse antibody against fascin
    Immunofluorescence double-staining to phenotype the P. acnes -positive cells in non-granulomatous areas of sarcoid lungs and lymph nodes. Green signal (FITC): PAB antibody; red signals (TRITC): <t>anti-CD68</t> antibody ( a , c , e ), <t>anti-fascin</t> antibody ( b , d , f ). Results of double-staining are merged in all pictures. In the sarcoid lung, all of the P. acnes signals overlapped (yellow) with CD68-positive alveolar macrophages ( a ), whereas the fascin-positive alveolar dendritic cells did not contain small round bodies detected by PAB antibody ( b ). In the paracortical area of the sarcoid lymph node, small round body detected by PAB antibody were found in the CD68-positive macrophages ( c ), but not in the fascin-positive dendritic cells ( d ). In the sinus of the sarcoid lymph node, Hamazaki-Wesenberg bodies detected by PAB antibody overlapped with CD68-positive macrophages ( e ), but not with the fascin-positive dendritic cells ( f ). Original magnification: a – b , × 400; c – f , × 1000.
    Monoclonal Mouse Antibody Against Fascin, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher fascin 1
    RSK2 activates CREB to promote cancer cell invasion and migration by up-regulating proinvasive <t>Fascin-1.</t> A , stable knockdown of RSK2 or CREB by shRNA ( lower panels ) results in significantly decreased invasion ( upper panels ) in the metastatic cell line
    Fascin 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc cell signaling antibody number
    RSK2 activates CREB to promote cancer cell invasion and migration by up-regulating proinvasive <t>Fascin-1.</t> A , stable knockdown of RSK2 or CREB by shRNA ( lower panels ) results in significantly decreased invasion ( upper panels ) in the metastatic cell line
    Cell Signaling Antibody Number, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies mouse anti fascin
    <t>Fascin</t> is upregulated in migrating neuroblasts. A , Immunostaining of sagittal brain sections shows strong fascin expression along the RMS in both P7 and adult mice. B , C , Confocal images from P7 mouse SVZ sections showing that fascin immunostaining virtually overlaps with Dcx+ migrating neuroblasts ( B ), but is excluded from <t>GFAP+</t> stem cells and astrocytes ( C ). D , Hardly any colocalization is observed with Mash1+ transit-amplifying progenitors. Scale bars: A , 500 μm; B–D , 10 μm.
    Mouse Anti Fascin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology anti fascin 1 mouse monoclonal antibody
    Resistin expression was associated with the survival of patients with colorectal cancer. (a, b). The associations of resistin expression with relapse-free survival (RFS) (a) and overall survival (OS) (b) were analyzed. (c, d). The associations of <t>fascin-1</t> expression with RFS (c) and OS (d) were analyzed. (e, f). Kaplan-Meier curves for OS (e) and RFS (f) of combined high expression of resistin and fascin-1 in colorectal cancer. P values were calculated using the Mantel-Cox log-rank test.
    Anti Fascin 1 Mouse Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher fascin
    Resistin expression was associated with the survival of patients with colorectal cancer. (a, b). The associations of resistin expression with relapse-free survival (RFS) (a) and overall survival (OS) (b) were analyzed. (c, d). The associations of <t>fascin-1</t> expression with RFS (c) and OS (d) were analyzed. (e, f). Kaplan-Meier curves for OS (e) and RFS (f) of combined high expression of resistin and fascin-1 in colorectal cancer. P values were calculated using the Mantel-Cox log-rank test.
    Fascin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies mouse anti human fascin
    TnTs express proteins characteristic of actin-based extensions. Confocal imaging demonstrated immunofluorescence of specific protein components of MSTO-211H cells with stained TnTs. a) Actin is uniform across the entirety of the TnT, which passes over an adherent cell. b) <t>Fascin</t> is expressed intermittently and at the base of nanotubes c) Pankeratin localizes to the perinuclear region of the cells. d) <t>Ezrin</t> expression was most prominent at the base of TnTs, consistent with its role in organizing actin-based filaments. e) ß-catenin is minimally present within TnTs. f) E-cadherin staining of TnTs between cells. g) ZO-1 localizes to the cell membrane, including the point of contact of TnTs. Scale bars: a) 20 µm, b) 20 µm, c) 30 µm, d) 30 µm, e) 20 µm, f) 20 µm, g) 50 µm.
    Mouse Anti Human Fascin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc plenti6 v5 dest fascin
    Validation and characterization of OS cell lines with altered <t>Fascin-1</t> expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a <t>pLenti6/V5-DEST</t> empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)
    Plenti6 V5 Dest Fascin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SIV is present in the rectal epithelium as early as four hours post infection. Serial paraffin sections of R-H4.1 (sacrificed four hours pi; A and B) and R-H16.2 (sacrificed sixteen hours pi; C and D) were labeled by IHF for gp130 (Alexa-546, A and C), CD3 (TRITC, B and D) or fascin (Alexa-488, B and D) and nuclei were stained with DAPI (A–D). CR, crypt; LP, lamina propria; RL, rectal lumen. Arrows on serial sections point to intraepithelial T lymphocytes positive for SIV antigens. Cell-associated virus in the epithelial cell fraction was assayed by co-culture of cells isolated from the rectum. Results are expressed as TCID 50 per million cells. The viral load does not increase over the first four days of infection; + the TCID 50 could not be calculated due to small number of wells positive for SIV antigen (E).

    Journal: PLoS ONE

    Article Title: Rapid Dissemination of SIV Follows Multisite Entry after Rectal Inoculation

    doi: 10.1371/journal.pone.0019493

    Figure Lengend Snippet: SIV is present in the rectal epithelium as early as four hours post infection. Serial paraffin sections of R-H4.1 (sacrificed four hours pi; A and B) and R-H16.2 (sacrificed sixteen hours pi; C and D) were labeled by IHF for gp130 (Alexa-546, A and C), CD3 (TRITC, B and D) or fascin (Alexa-488, B and D) and nuclei were stained with DAPI (A–D). CR, crypt; LP, lamina propria; RL, rectal lumen. Arrows on serial sections point to intraepithelial T lymphocytes positive for SIV antigens. Cell-associated virus in the epithelial cell fraction was assayed by co-culture of cells isolated from the rectum. Results are expressed as TCID 50 per million cells. The viral load does not increase over the first four days of infection; + the TCID 50 could not be calculated due to small number of wells positive for SIV antigen (E).

    Article Snippet: Primary antibodies were polyclonal rabbit anti-CD3 (DAKO, Trappes, France) mouse monoclonal antibodies to fascin (clone 55K-2, IgG1, DAKO), SIV gp130 envelope protein (clone KK46, IgG1, obtained from the NIH), SIV p27 capsid protein (IgG1, Advanced Biotechnologies Inc Columbia, MD), Aspergillus niger glucose oxidase (isotype control, IgG1, clone DAK-GO1, DAKO), HLA-DR (clone TÜ36, IgG2b, BD Biosciences), DC-SIGN (clone 120612, IgG2a, R & D Systems, Lille, France), DC-SIGN (clone 120507, IgG2b, R & D Systems), CD68 (clone KP1, IgG1, DAKO, Trappes, France), and tissue macrophage (clone PM-2K, IgG1, AbD Serotec, Düsseldorf, Germany).

    Techniques: Infection, Labeling, Immunohistofluorescence, Staining, Co-Culture Assay, Isolation

    SIV is present in lymphoid aggregates of the colo-rectal mucosa starting from four hours post infection. SIV DNA is amplified by nested PCR for gag in most lymphoid aggregates microdissected from paraffin sections of macaques sacrificed four hours to two days pi (A); ratios indicate the number of lymphoid aggregates positive for SIV DNA relative to the total number of lymphoid aggregates tested for each macaque. Clusters of SIV-antigen positive cells are observed in paraffin embedded sections of the colo-rectal mucosa of R-H4.1 (sacrificed four hours pi; B, C and D, serial sections, dashed circle indicates the same area in the three micrographs), R-H16.1 (sacrificed sixteen hours pi; E, F enlargement of area boxed in E), and R-H24.1 (sacrificed twenty-four hours pi; G, H enlargement of area boxed in G). These clusters of SIV + cells are not observed in serial sections of R-H16.1 (I, J enlargement of area boxed in I) or R-H24.1 (K, L enlargement of area boxed in K) labeled with an irrelevant antibody of the same isotype. These clusters contain T cells but not CD68 + macrophages or fascin + DCs. Sections were labeled by IHF for gp130 (Alexa-546, C), p27 (Alexa-488, E, F, G and H), CD3 (TRITC, B, D–H, K and L), fascin (Alexa-488, B), CD68 (Alexa-488, D), irrelevant antibody (Alexa-488 I–L) and nuclei were stained with DAPI (B–L). CR, crypt; LP, lamina propria; TA, T cell area of mucosal lymphoid aggregate; BF, B cell follicle.

    Journal: PLoS ONE

    Article Title: Rapid Dissemination of SIV Follows Multisite Entry after Rectal Inoculation

    doi: 10.1371/journal.pone.0019493

    Figure Lengend Snippet: SIV is present in lymphoid aggregates of the colo-rectal mucosa starting from four hours post infection. SIV DNA is amplified by nested PCR for gag in most lymphoid aggregates microdissected from paraffin sections of macaques sacrificed four hours to two days pi (A); ratios indicate the number of lymphoid aggregates positive for SIV DNA relative to the total number of lymphoid aggregates tested for each macaque. Clusters of SIV-antigen positive cells are observed in paraffin embedded sections of the colo-rectal mucosa of R-H4.1 (sacrificed four hours pi; B, C and D, serial sections, dashed circle indicates the same area in the three micrographs), R-H16.1 (sacrificed sixteen hours pi; E, F enlargement of area boxed in E), and R-H24.1 (sacrificed twenty-four hours pi; G, H enlargement of area boxed in G). These clusters of SIV + cells are not observed in serial sections of R-H16.1 (I, J enlargement of area boxed in I) or R-H24.1 (K, L enlargement of area boxed in K) labeled with an irrelevant antibody of the same isotype. These clusters contain T cells but not CD68 + macrophages or fascin + DCs. Sections were labeled by IHF for gp130 (Alexa-546, C), p27 (Alexa-488, E, F, G and H), CD3 (TRITC, B, D–H, K and L), fascin (Alexa-488, B), CD68 (Alexa-488, D), irrelevant antibody (Alexa-488 I–L) and nuclei were stained with DAPI (B–L). CR, crypt; LP, lamina propria; TA, T cell area of mucosal lymphoid aggregate; BF, B cell follicle.

    Article Snippet: Primary antibodies were polyclonal rabbit anti-CD3 (DAKO, Trappes, France) mouse monoclonal antibodies to fascin (clone 55K-2, IgG1, DAKO), SIV gp130 envelope protein (clone KK46, IgG1, obtained from the NIH), SIV p27 capsid protein (IgG1, Advanced Biotechnologies Inc Columbia, MD), Aspergillus niger glucose oxidase (isotype control, IgG1, clone DAK-GO1, DAKO), HLA-DR (clone TÜ36, IgG2b, BD Biosciences), DC-SIGN (clone 120612, IgG2a, R & D Systems, Lille, France), DC-SIGN (clone 120507, IgG2b, R & D Systems), CD68 (clone KP1, IgG1, DAKO, Trappes, France), and tissue macrophage (clone PM-2K, IgG1, AbD Serotec, Düsseldorf, Germany).

    Techniques: Infection, Amplification, Nested PCR, Labeling, Immunohistofluorescence, Staining

    Fascin localizes to comet tails in L. monocytogenes –infected cells. Infected BSC-1 cells (A and B) or XTC cells (C and D) were costained for actin (A and C) and fascin (B and D). Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex

    doi: 10.1083/jcb.200311040

    Figure Lengend Snippet: Fascin localizes to comet tails in L. monocytogenes –infected cells. Infected BSC-1 cells (A and B) or XTC cells (C and D) were costained for actin (A and C) and fascin (B and D). Bar, 10 μm.

    Article Snippet: For immunofluorescence, BSC-1 cells or XTC cells were cultured on polylysine-coated glass coverslips, infected with L. monocytogenes for 3 h, washed into media containing 50 μg/ml gentamicin, and incubated at 37°C for an additional 4 h. The cells were fixed in methanol and costained for fascin using an anti-fascin mAb (clone 55K-2; DakoCytomation), and for actin using an anti-actin rabbit pAb (Sigma-Aldrich).

    Techniques: Infection

    cbMg show cell clearance phenotype at the single cell, protein, and functional level (a) Schematic showing single nuclei RNA-seq (Fluidigm C1). Heatmap with unsupervised clustering displays the variation in the expression levels (z-score) of 75 most differentially expressed genes across all single nuclei. (b) Bar graphs show normalized expression levels (counts per million) of selected genes for single stMg/cbMg nuclei. Experiment was repeated independently 2 times with similar results. (c) Western blot analysis of CD11b-bead-isolated microglia (75,000 cbMg/stMg from one mouse per lane). Representative blots (left, cropped to show the specific band); quantifications (right). ACTB is used as loading control. FSCN1: stMg: mean=1.0, SEM=0.1887; cbMg: mean=0.1792, SEM=0.08138; p=0.0018, F=5.378, t 11 =3.994. MRC1: stMg: mean=1.0, SEM=0.2132; cbMg: mean=2.051, SEM=0.16; p=0.0043, F=1.776, t 8 =3.941. AXL: stMg: mean=1.0, SEM=0.3482; cbMg: mean=11.79, SEM=2.691; p=0.0018, F=59.71, t 12 =3.977. LC3: stMg: mean=1.0, SEM=0.2328; cbMg: mean=4.647, SEM=1.179; p=0.0162, F=25.66, t 8 . (d) Schematic showing phagocytosis assay of acutely isolated adult cbMg/stMg exposed to early apoptotic cells in vitro. Quantification of the percentage of GFP+ microglia that engulfed pHrodo+ early apoptotic cells in presence/absence of phagocytosis inhibitor, Cytochalasin D (Cyt) (stMg: mean=9.055, SEM=1.761; stMg+Cyt: mean= 0.5725, SEM=0.3420; cbMg: mean=24.63, SEM=0.3789; cbMg+Cyt: mean=2.720, SEM=1.589; p brain region

    Journal: Nature neuroscience

    Article Title: Epigenetic regulation of brain region-specific microglia clearance activity

    doi: 10.1038/s41593-018-0192-3

    Figure Lengend Snippet: cbMg show cell clearance phenotype at the single cell, protein, and functional level (a) Schematic showing single nuclei RNA-seq (Fluidigm C1). Heatmap with unsupervised clustering displays the variation in the expression levels (z-score) of 75 most differentially expressed genes across all single nuclei. (b) Bar graphs show normalized expression levels (counts per million) of selected genes for single stMg/cbMg nuclei. Experiment was repeated independently 2 times with similar results. (c) Western blot analysis of CD11b-bead-isolated microglia (75,000 cbMg/stMg from one mouse per lane). Representative blots (left, cropped to show the specific band); quantifications (right). ACTB is used as loading control. FSCN1: stMg: mean=1.0, SEM=0.1887; cbMg: mean=0.1792, SEM=0.08138; p=0.0018, F=5.378, t 11 =3.994. MRC1: stMg: mean=1.0, SEM=0.2132; cbMg: mean=2.051, SEM=0.16; p=0.0043, F=1.776, t 8 =3.941. AXL: stMg: mean=1.0, SEM=0.3482; cbMg: mean=11.79, SEM=2.691; p=0.0018, F=59.71, t 12 =3.977. LC3: stMg: mean=1.0, SEM=0.2328; cbMg: mean=4.647, SEM=1.179; p=0.0162, F=25.66, t 8 . (d) Schematic showing phagocytosis assay of acutely isolated adult cbMg/stMg exposed to early apoptotic cells in vitro. Quantification of the percentage of GFP+ microglia that engulfed pHrodo+ early apoptotic cells in presence/absence of phagocytosis inhibitor, Cytochalasin D (Cyt) (stMg: mean=9.055, SEM=1.761; stMg+Cyt: mean= 0.5725, SEM=0.3420; cbMg: mean=24.63, SEM=0.3789; cbMg+Cyt: mean=2.720, SEM=1.589; p brain region

    Article Snippet: Primary antibodies: AXL (1:500, sc-1097, Santa Cruz, or 1:1000, ab227871, Abcam) , FSCN1 (1:5000, ab126772, Abcam) , MRC1 (1:500, AF2535, R & D Systems) , LC3B (1:1000, 2775, Cell Signaling) , H3K27me3 (1:1000, 07-449, Millipore) , ACTB (1:20,000, ab8227, Abcam) , and Histone H3 (1:2,000, ab1791, Abcam) .

    Techniques: Functional Assay, RNA Sequencing Assay, Expressing, Western Blot, Isolation, Phagocytosis Assay, In Vitro

    The effect of fascin-1 overexpression on the reduction of cellular invasiveness by rapamycin. (a) Immunoblotting to determine the expression of fascin-1 and LC3-II. Actin was used as internal control. (b) Statistical analysis of the effect of fascin-1 overexpression on the change of cellular invasiveness brought upon by rapamycin treatment. ∗∗ p

    Journal: BioMed Research International

    Article Title: Autophagy Suppresses Invasiveness of Endometrial Cells through Reduction of Fascin-1

    doi: 10.1155/2018/8615435

    Figure Lengend Snippet: The effect of fascin-1 overexpression on the reduction of cellular invasiveness by rapamycin. (a) Immunoblotting to determine the expression of fascin-1 and LC3-II. Actin was used as internal control. (b) Statistical analysis of the effect of fascin-1 overexpression on the change of cellular invasiveness brought upon by rapamycin treatment. ∗∗ p

    Article Snippet: Antibodies against fascin-1 were from Abcam (Cambridge, USA).

    Techniques: Over Expression, Expressing

    Analysis of Cdc42 and fascin in MYC-nick–expressing cells. ( A ) MYC-nick–overexpressing HFF cells show increased filopodia formation that is further enhanced when Cdc42 is activated. (Magnification: 63×.) ( B ) HCT116 cells overexpressing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation

    doi: 10.1073/pnas.1610994113

    Figure Lengend Snippet: Analysis of Cdc42 and fascin in MYC-nick–expressing cells. ( A ) MYC-nick–overexpressing HFF cells show increased filopodia formation that is further enhanced when Cdc42 is activated. (Magnification: 63×.) ( B ) HCT116 cells overexpressing

    Article Snippet: Antibodies against c-MYC (9E10), Sin3, and fascin were from Santa Cruz Biotechnology.

    Techniques: Expressing

    Immunohistochemistry (IHC) of fascin and Cdc42 in human colon cancer biopsies. Representative IHC in normal mucosa, central area of the tumor, and the invasive front of the same tumor is shown. ( A – C ) Cdc42 IHC ( A ), fascin IHC ( B ), and MYC IHC

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation

    doi: 10.1073/pnas.1610994113

    Figure Lengend Snippet: Immunohistochemistry (IHC) of fascin and Cdc42 in human colon cancer biopsies. Representative IHC in normal mucosa, central area of the tumor, and the invasive front of the same tumor is shown. ( A – C ) Cdc42 IHC ( A ), fascin IHC ( B ), and MYC IHC

    Article Snippet: Antibodies against c-MYC (9E10), Sin3, and fascin were from Santa Cruz Biotechnology.

    Techniques: Immunohistochemistry

    Analysis of fascin expression in MYC-nick induced cell migration. ( A ) Effect of MYC-nick on abundance of endogenous fascin and exogenous GFP-fascin. ( B ) Fascin expression in murine intestinal and colonic lesions derived from different genetic backgrounds

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation

    doi: 10.1073/pnas.1610994113

    Figure Lengend Snippet: Analysis of fascin expression in MYC-nick induced cell migration. ( A ) Effect of MYC-nick on abundance of endogenous fascin and exogenous GFP-fascin. ( B ) Fascin expression in murine intestinal and colonic lesions derived from different genetic backgrounds

    Article Snippet: Antibodies against c-MYC (9E10), Sin3, and fascin were from Santa Cruz Biotechnology.

    Techniques: Expressing, Migration, Derivative Assay

    Stat3 and NFκB induce fascin expression in response to IL-6 and TNF-α. A , MDA-MB-231 cells were treated with TNF-α alone or IL-6 and TNF-α combined for 30 min, and ChIP analyses were performed with antibodies against Stat3, NFκB p50, or p65. NT, no treatment. B , quantitation of ChIP assays for MDA-MB-231 cells treated with IL-6 alone, TNF-α alone, or IL-6 and TNF-α combined for 30 min. ChIP analyses were performed for Stat3 or NFκB p50. Gels were quantitated using a phosphorimaging device and expressed as the intensities of ChIP/input with untreated values (NT) set at 1. Results represent the averages and S.D. of at least two experiments. C , cells were serum-starved for 48 h and treated with IL-6, TNF-α, or IL-6 plus TNF-α for 30 min or 2 h. RNA was extracted and subjected to quantitative real-time RT-PCR analyses for fascin. Results are standardized to GAPDH with untreated samples (NT) set at 1. Results represent the averages and S.D. of at least three independent experiements performed in triplicate.

    Journal: The Journal of Biological Chemistry

    Article Title: A Signal Transducer and Activator of Transcription 3·Nuclear Factor κB (Stat3·NFκB) Complex Is Necessary for the Expression of Fascin in Metastatic Breast Cancer Cells in Response to Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α *

    doi: 10.1074/jbc.M114.591719

    Figure Lengend Snippet: Stat3 and NFκB induce fascin expression in response to IL-6 and TNF-α. A , MDA-MB-231 cells were treated with TNF-α alone or IL-6 and TNF-α combined for 30 min, and ChIP analyses were performed with antibodies against Stat3, NFκB p50, or p65. NT, no treatment. B , quantitation of ChIP assays for MDA-MB-231 cells treated with IL-6 alone, TNF-α alone, or IL-6 and TNF-α combined for 30 min. ChIP analyses were performed for Stat3 or NFκB p50. Gels were quantitated using a phosphorimaging device and expressed as the intensities of ChIP/input with untreated values (NT) set at 1. Results represent the averages and S.D. of at least two experiments. C , cells were serum-starved for 48 h and treated with IL-6, TNF-α, or IL-6 plus TNF-α for 30 min or 2 h. RNA was extracted and subjected to quantitative real-time RT-PCR analyses for fascin. Results are standardized to GAPDH with untreated samples (NT) set at 1. Results represent the averages and S.D. of at least three independent experiements performed in triplicate.

    Article Snippet: Antibodies for Western blot analysis were anti-Stat3 (BD Transduction Laboratories), anti-tubulin (Sigma), anti- NFκB p50 and p65, and anti-fascin (Santa Cruz Biotechnology).

    Techniques: Expressing, Multiple Displacement Amplification, Chromatin Immunoprecipitation, Quantitation Assay, Quantitative RT-PCR

    NFκB p50 is required for fascin protein expression and for Stat3 binding to the fascin promoter. A , MDA-MB-231 cells were transfected with either a control siRNA or NFκBp50 siRNAs and cultured for 4 days. Western blot analyses were performed with antibodies against NFκB p50 or tubulin. B , two rounds of RNAi-mediated knockdown of NFκBp50 was performed (days 1 and 4). On day 7, cells were treated with TNF-α for 3 h followed by Western blot analyses. Autoradiographs of Western blotting analyses were quantitated by ImageJ software, and the results of three experiments + S.D. are shown. C , RNAi-mediated knockdown of NFκB p50 was performed as described in A . Cells were treated with IL-6 for 30 min. ChIP analyses were performed with antibodies against Stat3 or NFκB p50. Results represent two independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: A Signal Transducer and Activator of Transcription 3·Nuclear Factor κB (Stat3·NFκB) Complex Is Necessary for the Expression of Fascin in Metastatic Breast Cancer Cells in Response to Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α *

    doi: 10.1074/jbc.M114.591719

    Figure Lengend Snippet: NFκB p50 is required for fascin protein expression and for Stat3 binding to the fascin promoter. A , MDA-MB-231 cells were transfected with either a control siRNA or NFκBp50 siRNAs and cultured for 4 days. Western blot analyses were performed with antibodies against NFκB p50 or tubulin. B , two rounds of RNAi-mediated knockdown of NFκBp50 was performed (days 1 and 4). On day 7, cells were treated with TNF-α for 3 h followed by Western blot analyses. Autoradiographs of Western blotting analyses were quantitated by ImageJ software, and the results of three experiments + S.D. are shown. C , RNAi-mediated knockdown of NFκB p50 was performed as described in A . Cells were treated with IL-6 for 30 min. ChIP analyses were performed with antibodies against Stat3 or NFκB p50. Results represent two independent experiments.

    Article Snippet: Antibodies for Western blot analysis were anti-Stat3 (BD Transduction Laboratories), anti-tubulin (Sigma), anti- NFκB p50 and p65, and anti-fascin (Santa Cruz Biotechnology).

    Techniques: Expressing, Binding Assay, Multiple Displacement Amplification, Transfection, Cell Culture, Western Blot, Software, Chromatin Immunoprecipitation

    A 160-bp conserved region of the fascin promoter is sufficient to induce luciferase expression in response to IL-6. A , a 160-bp conserved region of the human fascin promoter containing an overlapping STAT site and NFκB site was subcloned into the luciferase reporter tkluc to generate FasP-luc. B , luciferase assays were performed in MDA-MB-231 cells transfected for 24 h with empty vector tkluc (negative control), M67 (positive control), which contains four Stat3 binding sites, and FasP-luc. Cells were treated with IL-6 for 6 h. C , MDA-MB-231 cells were transfected with tkluc or FasP-luc and treated with IL-6, TNF-α, or IL-6 and TNF-α together for 6 h. Results represent the averages and S.D. of at least three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: A Signal Transducer and Activator of Transcription 3·Nuclear Factor κB (Stat3·NFκB) Complex Is Necessary for the Expression of Fascin in Metastatic Breast Cancer Cells in Response to Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α *

    doi: 10.1074/jbc.M114.591719

    Figure Lengend Snippet: A 160-bp conserved region of the fascin promoter is sufficient to induce luciferase expression in response to IL-6. A , a 160-bp conserved region of the human fascin promoter containing an overlapping STAT site and NFκB site was subcloned into the luciferase reporter tkluc to generate FasP-luc. B , luciferase assays were performed in MDA-MB-231 cells transfected for 24 h with empty vector tkluc (negative control), M67 (positive control), which contains four Stat3 binding sites, and FasP-luc. Cells were treated with IL-6 for 6 h. C , MDA-MB-231 cells were transfected with tkluc or FasP-luc and treated with IL-6, TNF-α, or IL-6 and TNF-α together for 6 h. Results represent the averages and S.D. of at least three independent experiments.

    Article Snippet: Antibodies for Western blot analysis were anti-Stat3 (BD Transduction Laboratories), anti-tubulin (Sigma), anti- NFκB p50 and p65, and anti-fascin (Santa Cruz Biotechnology).

    Techniques: Luciferase, Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Negative Control, Positive Control, Binding Assay

    NFκB p65 is required for fascin expression. MDA-MB-231 cells were transfected with either a control siRNA or NFκBp65 siRNAs and cultured for 4 days. A , Western blot analyses of whole cell lysates were performed with antibodies against NFκB p65 or tubulin. B , cells were treated with TNF-α for 30 min followed by real-time RT-PCR analyses. C , cells were treated with TNF-α for 3 h followed by Western blot analyses. Autoradiographs of Western blotting analyses were quantitated by ImageJ software, and the results of three experiments + S.D. are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: A Signal Transducer and Activator of Transcription 3·Nuclear Factor κB (Stat3·NFκB) Complex Is Necessary for the Expression of Fascin in Metastatic Breast Cancer Cells in Response to Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α *

    doi: 10.1074/jbc.M114.591719

    Figure Lengend Snippet: NFκB p65 is required for fascin expression. MDA-MB-231 cells were transfected with either a control siRNA or NFκBp65 siRNAs and cultured for 4 days. A , Western blot analyses of whole cell lysates were performed with antibodies against NFκB p65 or tubulin. B , cells were treated with TNF-α for 30 min followed by real-time RT-PCR analyses. C , cells were treated with TNF-α for 3 h followed by Western blot analyses. Autoradiographs of Western blotting analyses were quantitated by ImageJ software, and the results of three experiments + S.D. are shown.

    Article Snippet: Antibodies for Western blot analysis were anti-Stat3 (BD Transduction Laboratories), anti-tubulin (Sigma), anti- NFκB p50 and p65, and anti-fascin (Santa Cruz Biotechnology).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Cell Culture, Western Blot, Quantitative RT-PCR, Software

    Phosphorylation of fascin-1 inhibits the formation of filopodia and phospho-S39-fascin-1 is dephosphorylated by mechanical stretch. ( A ) After isolation of phospho-proteins from S/US podocytes, a Western blot was performed. After staining for fascin-1 and phospho-S39-fascin-1, respectively, only a weak band was detected in stretched podocytes compared to unstretched podocytes. Blots of representative experiments of five independent experiments are shown. Protein load was checked by stain-free imaging. ( B ) 2-D gel image of S/US podocyte protein extract, which detects a shift of the fascin-1 protein spots to the alkaline pH-range (n = 3). The different fascin-1 spots (marked by arrowheads) were determined by LC-MS. ( C ) Podocytes were transfected with a plasmid encoding for wildtype-, non-phosphorylatable mutant eGFP-fascin-1-S39A (S39A) and the phosphomimetic mutant eGFP-fascin-1-S39D (S39D). Compared to wildtype and S39A-fascin-1, S39D-expressing podocytes were unable to develop filopodia neither under unstretched nor under stretched conditions. ( D ) Number of filopodia in wildtype-, S39A- and S39D-fascin-1 transfected cells after mechanical stress for 24 h. Error bars indicates SEM. ( E ) After transfection with a plasmid for eGFP-fascin-1-S39A, number of adherent cells after 3 days of mechanical stress is significantly higher than after transfection with eGFP-fascin-1-S39D. Bars with SEM; * p

    Journal: Scientific Reports

    Article Title: Studying the role of fascin-1 in mechanically stressed podocytes

    doi: 10.1038/s41598-017-10116-4

    Figure Lengend Snippet: Phosphorylation of fascin-1 inhibits the formation of filopodia and phospho-S39-fascin-1 is dephosphorylated by mechanical stretch. ( A ) After isolation of phospho-proteins from S/US podocytes, a Western blot was performed. After staining for fascin-1 and phospho-S39-fascin-1, respectively, only a weak band was detected in stretched podocytes compared to unstretched podocytes. Blots of representative experiments of five independent experiments are shown. Protein load was checked by stain-free imaging. ( B ) 2-D gel image of S/US podocyte protein extract, which detects a shift of the fascin-1 protein spots to the alkaline pH-range (n = 3). The different fascin-1 spots (marked by arrowheads) were determined by LC-MS. ( C ) Podocytes were transfected with a plasmid encoding for wildtype-, non-phosphorylatable mutant eGFP-fascin-1-S39A (S39A) and the phosphomimetic mutant eGFP-fascin-1-S39D (S39D). Compared to wildtype and S39A-fascin-1, S39D-expressing podocytes were unable to develop filopodia neither under unstretched nor under stretched conditions. ( D ) Number of filopodia in wildtype-, S39A- and S39D-fascin-1 transfected cells after mechanical stress for 24 h. Error bars indicates SEM. ( E ) After transfection with a plasmid for eGFP-fascin-1-S39A, number of adherent cells after 3 days of mechanical stress is significantly higher than after transfection with eGFP-fascin-1-S39D. Bars with SEM; * p

    Article Snippet: Membranes were immersed overnight in blocking buffer (10 mM Tris, 100 mM NaCl, 5% non-fat dry milk, 0.2% Tween-20, pH 7.5) and incubated for 2 h at room temperature in TBS-Tween (0.2%) with the following antibodies: anti-fascin-1 (sc-21743, Santa Cruz), anti-fascin-1 Ser-39-phospho-specific antibody (FP2661; ECM Biosciences, Versailles, KY, USA) and anti-Gapdh (sc-25778, Santa Cruz).

    Techniques: Isolation, Western Blot, Staining, Imaging, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Mutagenesis, Expressing

    Fascin-1 expression in response to mechanical stretch. ( A ) Quantitative RT-PCR revealed no significant differences in fascin-1 mRNA expression in S/US podocytes (n = 3). ( B ) Western blot showed no difference in the expression for fascin-1 in S/US podocytes. Relative fascin-1 protein levels on Western blots were quantified for unstretched (grey bar) to stretched cells (white bar) and revealed only a slightly but not significant down-regulation of fascin-1 after mechanical stress (n = 5). ( C ) LC-MS analysis of fascin-1 protein level (n = 4). ( D ) Immunofluorescence colocalization analysis of fascin-1 (red) and F-actin (green). Unstretched podocytes showed transversal actin fibers which reorganized into radial actin fibers converging into an ARC due to mechanical stress. In stretched podocytes the fluorescence intensity of the actin staining decreases anterogradely starting from the centre, whereas the intensity of the fascin-1 staining increases as depicted in the merged picture. ( E ) The quantitative analysis of the fluorescence intensities of stretched filaments is shown in the histogram. For quantification, the fluorescence intensities of F-actin and fascin-1 along single stress fibers (n = 5) were measured in 15 representative stretched cells starting from the actin rich-center (ARC) to the periphery of the cell. Experiments were normalized to Rpl32 mRNA ( A ) or Gapdh protein levels ( B , C ). Stretch parameters: cycle frequency of 0.5 Hz, linear strain of 5% for 3 days. Data are presented as means ± SEM. Scale bars represent 25 µm.

    Journal: Scientific Reports

    Article Title: Studying the role of fascin-1 in mechanically stressed podocytes

    doi: 10.1038/s41598-017-10116-4

    Figure Lengend Snippet: Fascin-1 expression in response to mechanical stretch. ( A ) Quantitative RT-PCR revealed no significant differences in fascin-1 mRNA expression in S/US podocytes (n = 3). ( B ) Western blot showed no difference in the expression for fascin-1 in S/US podocytes. Relative fascin-1 protein levels on Western blots were quantified for unstretched (grey bar) to stretched cells (white bar) and revealed only a slightly but not significant down-regulation of fascin-1 after mechanical stress (n = 5). ( C ) LC-MS analysis of fascin-1 protein level (n = 4). ( D ) Immunofluorescence colocalization analysis of fascin-1 (red) and F-actin (green). Unstretched podocytes showed transversal actin fibers which reorganized into radial actin fibers converging into an ARC due to mechanical stress. In stretched podocytes the fluorescence intensity of the actin staining decreases anterogradely starting from the centre, whereas the intensity of the fascin-1 staining increases as depicted in the merged picture. ( E ) The quantitative analysis of the fluorescence intensities of stretched filaments is shown in the histogram. For quantification, the fluorescence intensities of F-actin and fascin-1 along single stress fibers (n = 5) were measured in 15 representative stretched cells starting from the actin rich-center (ARC) to the periphery of the cell. Experiments were normalized to Rpl32 mRNA ( A ) or Gapdh protein levels ( B , C ). Stretch parameters: cycle frequency of 0.5 Hz, linear strain of 5% for 3 days. Data are presented as means ± SEM. Scale bars represent 25 µm.

    Article Snippet: Membranes were immersed overnight in blocking buffer (10 mM Tris, 100 mM NaCl, 5% non-fat dry milk, 0.2% Tween-20, pH 7.5) and incubated for 2 h at room temperature in TBS-Tween (0.2%) with the following antibodies: anti-fascin-1 (sc-21743, Santa Cruz), anti-fascin-1 Ser-39-phospho-specific antibody (FP2661; ECM Biosciences, Versailles, KY, USA) and anti-Gapdh (sc-25778, Santa Cruz).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Fluorescence, Staining

    Knockdown of fascin-1 influences the morphology of stretched podocytes and reduces the number of focal adhesions. ( A ) Immunofluorescence staining of fascin-1 (red) and F-actin (green) of control and fascin-1 KD cells after 3 days of biaxial mechanical stress with 0.5 Hz and 5% elongation. ( B ) Verification of fascin-1 KD by Western blot. ( C ) Podocytes adhesion after 3 days of stretch (n = 3). ( D ) Structured illumination microscopy (SIM) images showed co-localization of fascin-1 (green), talin-1 (red) and F-actin (white) in cultured podocytes. ( E ) Immunofluorescence staining of fascin-1 (green) and talin-1 (red) of control and fascin-1 KD cells. Fascin-1 KD (cells marked with arrowheads) reduced the number of talin 1-positive focal adhesions. Control podocytes (marked by arrows) showed more and thicker talin-1 contacts. (F-H) Quantitative analysis of focal adhesions (talin-1) in control (n = 185) and fascin-1 KD cells (n = 280). ( F ) Number of focal adhesions (FAs) per cell was reduced by 36 ± 2% after the KD of fascin-1 (125 ± 3 focal contacts per cell) compared to control transfected podocytes (196 ± 7 focal contacts per cell), p = 2.14E-22. ( G ) Area size of each FA per cell was reduced by 19.6 ± 1.1 % after fascin-1 KD (Ctrl: 1.48 ± 0.04 µm 2 , KD: 1.19 ± 0.02 µm 2 , p = 1.95E-14). ( H ) Area size of total FAs per cell showed a reduction by 49.5 ± 1.6% in fascin-1 KD podocytes (Ctrl: 304 ± 16 µm 2 , KD: 154 ± 5 µm 2 , p = 1.72E-22). Error bars indicate SEM; * p

    Journal: Scientific Reports

    Article Title: Studying the role of fascin-1 in mechanically stressed podocytes

    doi: 10.1038/s41598-017-10116-4

    Figure Lengend Snippet: Knockdown of fascin-1 influences the morphology of stretched podocytes and reduces the number of focal adhesions. ( A ) Immunofluorescence staining of fascin-1 (red) and F-actin (green) of control and fascin-1 KD cells after 3 days of biaxial mechanical stress with 0.5 Hz and 5% elongation. ( B ) Verification of fascin-1 KD by Western blot. ( C ) Podocytes adhesion after 3 days of stretch (n = 3). ( D ) Structured illumination microscopy (SIM) images showed co-localization of fascin-1 (green), talin-1 (red) and F-actin (white) in cultured podocytes. ( E ) Immunofluorescence staining of fascin-1 (green) and talin-1 (red) of control and fascin-1 KD cells. Fascin-1 KD (cells marked with arrowheads) reduced the number of talin 1-positive focal adhesions. Control podocytes (marked by arrows) showed more and thicker talin-1 contacts. (F-H) Quantitative analysis of focal adhesions (talin-1) in control (n = 185) and fascin-1 KD cells (n = 280). ( F ) Number of focal adhesions (FAs) per cell was reduced by 36 ± 2% after the KD of fascin-1 (125 ± 3 focal contacts per cell) compared to control transfected podocytes (196 ± 7 focal contacts per cell), p = 2.14E-22. ( G ) Area size of each FA per cell was reduced by 19.6 ± 1.1 % after fascin-1 KD (Ctrl: 1.48 ± 0.04 µm 2 , KD: 1.19 ± 0.02 µm 2 , p = 1.95E-14). ( H ) Area size of total FAs per cell showed a reduction by 49.5 ± 1.6% in fascin-1 KD podocytes (Ctrl: 304 ± 16 µm 2 , KD: 154 ± 5 µm 2 , p = 1.72E-22). Error bars indicate SEM; * p

    Article Snippet: Membranes were immersed overnight in blocking buffer (10 mM Tris, 100 mM NaCl, 5% non-fat dry milk, 0.2% Tween-20, pH 7.5) and incubated for 2 h at room temperature in TBS-Tween (0.2%) with the following antibodies: anti-fascin-1 (sc-21743, Santa Cruz), anti-fascin-1 Ser-39-phospho-specific antibody (FP2661; ECM Biosciences, Versailles, KY, USA) and anti-Gapdh (sc-25778, Santa Cruz).

    Techniques: Immunofluorescence, Staining, Western Blot, Microscopy, Cell Culture, Transfection

    Effect of S39A-fascin-1, S39D-fascin-1 and wildtype fascin-1 on cell spreading and filopodia formation during mechanical stress. ( A ) Confluent podocytes were transfected with eGFP-fascin-1 wildtype (WT), eGFP-fascin-1-S39A and eGFP-fascin-1-S39D and Ca 2+ -depleted for 30 min. After re-addition of Ca 2+ , the cells were mechanical stretched. Cell spreading and filopodia formation occurred within 60 min and was completed after 90 min in control cells as well as in podocytes expressing eGFP-fascin-1-S39A. In contrast, eGFP-fascin-1-S39D transfected podocytes showed a poor spreading and no filopodia formation during 90 min. Scale bar represent 50 µm. ( B ) Quantitative analysis showed that 95 ± 3% of wildtype cells and 106 ± 14% of S39A-fascin-1 transfected cells were still adherent after 90 min in the presence of mechanical stress. In contrast, the number of S39D-fascin-1 transfected podocytes significantly decreased to 64 ± 13%. Data are mean ± SD of n = 4 experiments, ** p

    Journal: Scientific Reports

    Article Title: Studying the role of fascin-1 in mechanically stressed podocytes

    doi: 10.1038/s41598-017-10116-4

    Figure Lengend Snippet: Effect of S39A-fascin-1, S39D-fascin-1 and wildtype fascin-1 on cell spreading and filopodia formation during mechanical stress. ( A ) Confluent podocytes were transfected with eGFP-fascin-1 wildtype (WT), eGFP-fascin-1-S39A and eGFP-fascin-1-S39D and Ca 2+ -depleted for 30 min. After re-addition of Ca 2+ , the cells were mechanical stretched. Cell spreading and filopodia formation occurred within 60 min and was completed after 90 min in control cells as well as in podocytes expressing eGFP-fascin-1-S39A. In contrast, eGFP-fascin-1-S39D transfected podocytes showed a poor spreading and no filopodia formation during 90 min. Scale bar represent 50 µm. ( B ) Quantitative analysis showed that 95 ± 3% of wildtype cells and 106 ± 14% of S39A-fascin-1 transfected cells were still adherent after 90 min in the presence of mechanical stress. In contrast, the number of S39D-fascin-1 transfected podocytes significantly decreased to 64 ± 13%. Data are mean ± SD of n = 4 experiments, ** p

    Article Snippet: Membranes were immersed overnight in blocking buffer (10 mM Tris, 100 mM NaCl, 5% non-fat dry milk, 0.2% Tween-20, pH 7.5) and incubated for 2 h at room temperature in TBS-Tween (0.2%) with the following antibodies: anti-fascin-1 (sc-21743, Santa Cruz), anti-fascin-1 Ser-39-phospho-specific antibody (FP2661; ECM Biosciences, Versailles, KY, USA) and anti-Gapdh (sc-25778, Santa Cruz).

    Techniques: Transfection, Expressing

    Fascin-1 is predominantly expressed in mouse podocytes in vivo . ( A ) Kidney sections were stained for fascin-1 (green) and nephrin (red) (I,II). By superresolution microscopy SIM, single podocytes were visualized expressing fascin-1 in their major processes (III, green). Nuclei were stained with Hoechst (blue). Scale bars represent 25 µm (I) and 10 µm (II, III), respectively. ( B ) Quantitative measurement of fascin-1 mRNA isolated of primary podocytes, total kidney, glomeruli and brain as a control. ( C ) Western blot of the expression of fascin-1 isolated from total kidney, glomeruli, primary podocytes and brain (as a positive control). Values were normalized with Gapdh for qRT-PCR and β-actin for Western blot analysis. Data are presented as means ± SEM; * p

    Journal: Scientific Reports

    Article Title: Studying the role of fascin-1 in mechanically stressed podocytes

    doi: 10.1038/s41598-017-10116-4

    Figure Lengend Snippet: Fascin-1 is predominantly expressed in mouse podocytes in vivo . ( A ) Kidney sections were stained for fascin-1 (green) and nephrin (red) (I,II). By superresolution microscopy SIM, single podocytes were visualized expressing fascin-1 in their major processes (III, green). Nuclei were stained with Hoechst (blue). Scale bars represent 25 µm (I) and 10 µm (II, III), respectively. ( B ) Quantitative measurement of fascin-1 mRNA isolated of primary podocytes, total kidney, glomeruli and brain as a control. ( C ) Western blot of the expression of fascin-1 isolated from total kidney, glomeruli, primary podocytes and brain (as a positive control). Values were normalized with Gapdh for qRT-PCR and β-actin for Western blot analysis. Data are presented as means ± SEM; * p

    Article Snippet: Membranes were immersed overnight in blocking buffer (10 mM Tris, 100 mM NaCl, 5% non-fat dry milk, 0.2% Tween-20, pH 7.5) and incubated for 2 h at room temperature in TBS-Tween (0.2%) with the following antibodies: anti-fascin-1 (sc-21743, Santa Cruz), anti-fascin-1 Ser-39-phospho-specific antibody (FP2661; ECM Biosciences, Versailles, KY, USA) and anti-Gapdh (sc-25778, Santa Cruz).

    Techniques: In Vivo, Staining, Microscopy, Expressing, Isolation, Western Blot, Positive Control, Quantitative RT-PCR

    Kidney biopsies of patients suffering from diabetic nephropathy (DN) have a reduced phospho-S39-fascin-1 expression. ( A ) Expression of fascin-1 (green) and nephrin (red) in human kidneys taken by SIM. Podocytes are marked with arrowheads. ( B ) Expression of fascin-1 is not reduced by patients suffering from DN. ( C ) Expression of phospho-S39-fascin-1 was reduced in glomeruli of patients suffering from DN compared to control tissue. Scale bars represent 10 µm ( A ) and 50 µm ( B , C ), respectively.

    Journal: Scientific Reports

    Article Title: Studying the role of fascin-1 in mechanically stressed podocytes

    doi: 10.1038/s41598-017-10116-4

    Figure Lengend Snippet: Kidney biopsies of patients suffering from diabetic nephropathy (DN) have a reduced phospho-S39-fascin-1 expression. ( A ) Expression of fascin-1 (green) and nephrin (red) in human kidneys taken by SIM. Podocytes are marked with arrowheads. ( B ) Expression of fascin-1 is not reduced by patients suffering from DN. ( C ) Expression of phospho-S39-fascin-1 was reduced in glomeruli of patients suffering from DN compared to control tissue. Scale bars represent 10 µm ( A ) and 50 µm ( B , C ), respectively.

    Article Snippet: Membranes were immersed overnight in blocking buffer (10 mM Tris, 100 mM NaCl, 5% non-fat dry milk, 0.2% Tween-20, pH 7.5) and incubated for 2 h at room temperature in TBS-Tween (0.2%) with the following antibodies: anti-fascin-1 (sc-21743, Santa Cruz), anti-fascin-1 Ser-39-phospho-specific antibody (FP2661; ECM Biosciences, Versailles, KY, USA) and anti-Gapdh (sc-25778, Santa Cruz).

    Techniques: Expressing

    Fascin colocalizes and physically interacts with Pol2 inside the nucleus. ( A ) BT-20 cells were grown on coverlips, fixed and stained for confocal microscopy. Primary antibodies against pFascin (red) and Pol2 (green) were used followed by visualization using Alexa-conjugated secondary antibodies. The arrows point to colocalizing clusters. Top right hand panel: Scatter plot of Fascin and Pol2 intensities. Top right corner: Calculated pearson correlation coefficient. Scale bar, 10 μm. -Ab: primary antibodies omitted. ( B ) BT-20 nuclear protein extracts were fractionated using size exclusion chromatography followed by western blot analysis using the indicated Pol2 and pFascin antibodies. Arrows point to fractions where Pol2 and pFascin co-elute.

    Journal: Scientific Reports

    Article Title: Insights into a novel nuclear function for Fascin in the regulation of the amino-acid transporter SLC3A2

    doi: 10.1038/srep36699

    Figure Lengend Snippet: Fascin colocalizes and physically interacts with Pol2 inside the nucleus. ( A ) BT-20 cells were grown on coverlips, fixed and stained for confocal microscopy. Primary antibodies against pFascin (red) and Pol2 (green) were used followed by visualization using Alexa-conjugated secondary antibodies. The arrows point to colocalizing clusters. Top right hand panel: Scatter plot of Fascin and Pol2 intensities. Top right corner: Calculated pearson correlation coefficient. Scale bar, 10 μm. -Ab: primary antibodies omitted. ( B ) BT-20 nuclear protein extracts were fractionated using size exclusion chromatography followed by western blot analysis using the indicated Pol2 and pFascin antibodies. Arrows point to fractions where Pol2 and pFascin co-elute.

    Article Snippet: Antibodies targeting Fascin (Santa cruz, sc-21743, 1:200), pFascin (ECM biosciences, FP-2661, 1:1000), Pol2 (Covance, 8WG16, 1:200), mTOR (Cell signalling technologies, 2983, 1:500) were then added directly to the blocking buffer for 1 hour.

    Techniques: Staining, Confocal Microscopy, Size-exclusion Chromatography, Western Blot

    ChIP-seq analysis reveals remarkable colocalization of pFascin with H3K4me3. ( A ) Histogram representing pFascin binding sites distribution and density relative to H3K4me3 peaks. ( B ) Histogram representing pFascin and H3K4me3 binding sites distribution and density relative to the transcription start site (TSS). ( C ) The interaction of Fascin protein with H3K4 methylatransferase complex was analyzed following immunoprecipitation of BT-20 extracts with antibodies directed against pFascin followed by western blot analysis and immunochemical detection of RbBP5 and pFascin. ( D ) Screenshot from Scaffold 4 program showing the results of mass spectrometry data of H3K4me3 core subunits interactors. Fascin is clearly and specifically interacting with RbBP5. For each subunit, the immunoprecipitation was done in triplicate. The percentages represent the peptide identification probability.

    Journal: Scientific Reports

    Article Title: Insights into a novel nuclear function for Fascin in the regulation of the amino-acid transporter SLC3A2

    doi: 10.1038/srep36699

    Figure Lengend Snippet: ChIP-seq analysis reveals remarkable colocalization of pFascin with H3K4me3. ( A ) Histogram representing pFascin binding sites distribution and density relative to H3K4me3 peaks. ( B ) Histogram representing pFascin and H3K4me3 binding sites distribution and density relative to the transcription start site (TSS). ( C ) The interaction of Fascin protein with H3K4 methylatransferase complex was analyzed following immunoprecipitation of BT-20 extracts with antibodies directed against pFascin followed by western blot analysis and immunochemical detection of RbBP5 and pFascin. ( D ) Screenshot from Scaffold 4 program showing the results of mass spectrometry data of H3K4me3 core subunits interactors. Fascin is clearly and specifically interacting with RbBP5. For each subunit, the immunoprecipitation was done in triplicate. The percentages represent the peptide identification probability.

    Article Snippet: Antibodies targeting Fascin (Santa cruz, sc-21743, 1:200), pFascin (ECM biosciences, FP-2661, 1:1000), Pol2 (Covance, 8WG16, 1:200), mTOR (Cell signalling technologies, 2983, 1:500) were then added directly to the blocking buffer for 1 hour.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Western Blot, Mass Spectrometry

    Fascin localizes to the nucleus in both breast cancer cell lines and tissue. Fascin and pFascin show similar localization in vitro and in vivo . ( A ) BT-20 and MDA-MB-231 cells were grown on coverlips, fixed and stained for confocal microscopy. Primary antibodies against Fascin (green) and pFascin (red) were used followed by visualization using Alexa-conjugated secondary antibodies. Punctate staining corresponding to both Fascin isoforms is visible inside the nucleus. Scale bar, 10 μm. -Ab: primary antibodies omitted. ( B ) Cytoplasmic and nuclear extracts were prepared from WT and Fascin knockdown BT-20 cells and immunoblotted for Fascin and pFascin with corresponding antibodies. GAPDH and Lamin were used as fractionation controls. ( C ) Representative images of immunohistochemical staining of Fascin and pFascin in a multi-tissueTMA. Magnification: 40x. ( D , E ) Distribution of Fascin and pFascin in nuclear and cytoplasmic fractions analyzed by immunoblotting. ( D–F ) Fascin and P-Fascin expression across different stages of breast cancer progression. ( D ) Representative images of Fascin and P-Fascin expression in benign tissue (BN) and breast cancer (CA). ( E ) Fascin and P-Fascin protein expression at different stages of breast cancer progression and ( F ) lymph nodes status. Confidence intervals (95%) show normalized mean intensity value units of Fascin and P-Fascin as determined by quantitative evaluation of immunohistochemistry. The y-axis represents numerical values corresponding to the intensity of Fascin and P-Fascin expression (0, negative; 1, weak; 2, moderate and 3, high intensity). pN0: lymph nodes pathologically negative and pN+: lymph nodes pathologically positive.

    Journal: Scientific Reports

    Article Title: Insights into a novel nuclear function for Fascin in the regulation of the amino-acid transporter SLC3A2

    doi: 10.1038/srep36699

    Figure Lengend Snippet: Fascin localizes to the nucleus in both breast cancer cell lines and tissue. Fascin and pFascin show similar localization in vitro and in vivo . ( A ) BT-20 and MDA-MB-231 cells were grown on coverlips, fixed and stained for confocal microscopy. Primary antibodies against Fascin (green) and pFascin (red) were used followed by visualization using Alexa-conjugated secondary antibodies. Punctate staining corresponding to both Fascin isoforms is visible inside the nucleus. Scale bar, 10 μm. -Ab: primary antibodies omitted. ( B ) Cytoplasmic and nuclear extracts were prepared from WT and Fascin knockdown BT-20 cells and immunoblotted for Fascin and pFascin with corresponding antibodies. GAPDH and Lamin were used as fractionation controls. ( C ) Representative images of immunohistochemical staining of Fascin and pFascin in a multi-tissueTMA. Magnification: 40x. ( D , E ) Distribution of Fascin and pFascin in nuclear and cytoplasmic fractions analyzed by immunoblotting. ( D–F ) Fascin and P-Fascin expression across different stages of breast cancer progression. ( D ) Representative images of Fascin and P-Fascin expression in benign tissue (BN) and breast cancer (CA). ( E ) Fascin and P-Fascin protein expression at different stages of breast cancer progression and ( F ) lymph nodes status. Confidence intervals (95%) show normalized mean intensity value units of Fascin and P-Fascin as determined by quantitative evaluation of immunohistochemistry. The y-axis represents numerical values corresponding to the intensity of Fascin and P-Fascin expression (0, negative; 1, weak; 2, moderate and 3, high intensity). pN0: lymph nodes pathologically negative and pN+: lymph nodes pathologically positive.

    Article Snippet: Antibodies targeting Fascin (Santa cruz, sc-21743, 1:200), pFascin (ECM biosciences, FP-2661, 1:1000), Pol2 (Covance, 8WG16, 1:200), mTOR (Cell signalling technologies, 2983, 1:500) were then added directly to the blocking buffer for 1 hour.

    Techniques: In Vitro, In Vivo, Multiple Displacement Amplification, Staining, Confocal Microscopy, Fractionation, Immunohistochemistry, Expressing

    Induction of STAT3, FASCIN and SURVIVIN expression by c-SRC and suppression by MLT in MCF-7 breast cancer cells. MCF-7 breast cancer cells stably transfected with the constitutively active SRC-D construct and treated with MLT (10 −9 M or 10 −8 M) for 24 h, after which the expression of t-STAT3, pSTAT3, t-FASCIN, and t-SURVIVIN levels were measured by Western blot. β-actin was used as a control for equal loading.

    Journal: Journal of pineal research

    Article Title: Epigenetic inhibition of the tumor suppressor ARHI by light at night-induced circadian melatonin disruption mediates STAT3-driven paclitaxel resistance in breast cancer

    doi: 10.1111/jpi.12586

    Figure Lengend Snippet: Induction of STAT3, FASCIN and SURVIVIN expression by c-SRC and suppression by MLT in MCF-7 breast cancer cells. MCF-7 breast cancer cells stably transfected with the constitutively active SRC-D construct and treated with MLT (10 −9 M or 10 −8 M) for 24 h, after which the expression of t-STAT3, pSTAT3, t-FASCIN, and t-SURVIVIN levels were measured by Western blot. β-actin was used as a control for equal loading.

    Article Snippet: Blots were probed with various antibodies including (t) total and phospho (p)-HER2, HER3, ERK1/2 Thr202/Tyr204, ERK1/2, AKT Ser473, AKT, SRC Ser536, SRC, CREB Ser133, CREB, RAS, STAT3 Tyr707, Acetyl-STAT3 (K685) and STAT3, SURVIVIN, and FASCIN from Cell Signaling (Danvers, MA).

    Techniques: Expressing, Stable Transfection, Transfection, Construct, Western Blot

    Evaluation of STAT3, FASCIN and SURVIVIN expression, and histologic staining in breast tumor xenografts in response to dLAN, MLT, and PTX. Modulation of the STAT3 transcription factor and its downstream targets FASCIN and SURVIVIN in tissue-isolated (ERα+) MCF-7 human breast tumor xenografts from female nude rats housed in a dLAN photoperiod and treated with vehicle (dLAN), PTX (dLAN + PTX), MLT during dLAN (dLAN + MLT), MLT plus PTX during dLAN (dLAN + MLT + PTX) or a LD, 12:12 lighting schedule and treated with vehicle (LD, 12:12) or PTX (LD 12:12 + PTX). ( A ) Western blot of total (t) and phospho (p) STAT3, FASCIN, and t-SURVIVN from total tissue lysates of breast tumor xenografts harvested from rats in each group above. All tumors were harvested at 2400 hr (mid-dLAN phase) from 3 animals in each group. Total cell lysates (120 μg of protein per sample) from each tumor were analyzed by Western blot. β-actin was used as a control for equal loading. ( B ) Characterization of cytoplasmic and nuclear staining of STAT3 in MCF-7 tissue-isolated breast tumor xenografts from dLAN, dLAN + PTX, dLAN + MLT, and dLAN + MLT + PTX treatment groups. Representative IHC of tumor xenografts using anti-STAT3 (Cell Signaling) (1000 x magnification).

    Journal: Journal of pineal research

    Article Title: Epigenetic inhibition of the tumor suppressor ARHI by light at night-induced circadian melatonin disruption mediates STAT3-driven paclitaxel resistance in breast cancer

    doi: 10.1111/jpi.12586

    Figure Lengend Snippet: Evaluation of STAT3, FASCIN and SURVIVIN expression, and histologic staining in breast tumor xenografts in response to dLAN, MLT, and PTX. Modulation of the STAT3 transcription factor and its downstream targets FASCIN and SURVIVIN in tissue-isolated (ERα+) MCF-7 human breast tumor xenografts from female nude rats housed in a dLAN photoperiod and treated with vehicle (dLAN), PTX (dLAN + PTX), MLT during dLAN (dLAN + MLT), MLT plus PTX during dLAN (dLAN + MLT + PTX) or a LD, 12:12 lighting schedule and treated with vehicle (LD, 12:12) or PTX (LD 12:12 + PTX). ( A ) Western blot of total (t) and phospho (p) STAT3, FASCIN, and t-SURVIVN from total tissue lysates of breast tumor xenografts harvested from rats in each group above. All tumors were harvested at 2400 hr (mid-dLAN phase) from 3 animals in each group. Total cell lysates (120 μg of protein per sample) from each tumor were analyzed by Western blot. β-actin was used as a control for equal loading. ( B ) Characterization of cytoplasmic and nuclear staining of STAT3 in MCF-7 tissue-isolated breast tumor xenografts from dLAN, dLAN + PTX, dLAN + MLT, and dLAN + MLT + PTX treatment groups. Representative IHC of tumor xenografts using anti-STAT3 (Cell Signaling) (1000 x magnification).

    Article Snippet: Blots were probed with various antibodies including (t) total and phospho (p)-HER2, HER3, ERK1/2 Thr202/Tyr204, ERK1/2, AKT Ser473, AKT, SRC Ser536, SRC, CREB Ser133, CREB, RAS, STAT3 Tyr707, Acetyl-STAT3 (K685) and STAT3, SURVIVIN, and FASCIN from Cell Signaling (Danvers, MA).

    Techniques: Expressing, Staining, Isolation, Western Blot, Immunohistochemistry

    Model of the functions of Jumu in filopodium formation and circulating hemocyte phagocytosis. a Normal expression of Jumu maintains the levels of NimC1, Enabled and Fascin in a direct or indirect manner. Enabled and Fascin participate in filopodium formation. The phagocytosis receptor NimC1 is involved in the recognition of pathogens and the subcellular localization of Ena. b Deficiency of Jumu reduces the protein levels of NimC1, Enabled and Fascin and consequently inhibits filopodium formation and hemocyte phagocytosis. c Severe deficiency of jumu can inhibit normal hemocyte mitosis and result in enlarged multinucleated hemocytes. The loss of jumu may control the cell cycle and mitosis process by affecting the expression of the Cyclin genes png and piwi . Severe deficiency of jumu also induces the generation of lamellocytes through the activation of the Toll signaling pathway

    Journal: Cell Communication and Signaling : CCS

    Article Title: Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila

    doi: 10.1186/s12964-018-0305-3

    Figure Lengend Snippet: Model of the functions of Jumu in filopodium formation and circulating hemocyte phagocytosis. a Normal expression of Jumu maintains the levels of NimC1, Enabled and Fascin in a direct or indirect manner. Enabled and Fascin participate in filopodium formation. The phagocytosis receptor NimC1 is involved in the recognition of pathogens and the subcellular localization of Ena. b Deficiency of Jumu reduces the protein levels of NimC1, Enabled and Fascin and consequently inhibits filopodium formation and hemocyte phagocytosis. c Severe deficiency of jumu can inhibit normal hemocyte mitosis and result in enlarged multinucleated hemocytes. The loss of jumu may control the cell cycle and mitosis process by affecting the expression of the Cyclin genes png and piwi . Severe deficiency of jumu also induces the generation of lamellocytes through the activation of the Toll signaling pathway

    Article Snippet: Then, 30 μg of the lysate was loaded into a 12% SDS-PAGE gel, followed by electroblotting onto nitrocellulose membranes and probing with mouse anti-α-tubulin (1:500, Sigma), mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (1:300, Developmental Studies Hybridoma Bank) for 2 h. The blot was subsequently probed with anti-mouse HRP-conjugated secondary antibodies for 1.5 h and detected using the ECL Plus detection system (Program).

    Techniques: Expressing, Activation Assay

    Overexpression of jumu induces cell spreading and large numbers of filopodia in hemocytes. a-d Phalloidin staining (green) shows that Hml > GFP > UAS-jumu displays an increased number of filopodia and larger lamellipodia than the control ( a and b ); knockdown of ena or fascin in Hml > GFP > UAS-jumu can inhibit cell spreading and the formation of numerous filopodia ( c and d ). e-g Quantification of filopodium numbers and lengths and lamellipodial area. h Quantification of the phagocytosis indexes for latex beads, B. bassiana , S. aureus or E. coli. i-l Quantification of Ena, Fascin, Rho1 and Profilin levels. m Western blot analysis using antibodies against Ena, Fascin, Profilin and Rho1 and the corresponding graphical representation show that the levels of Ena and Fascin are increased in jumu -overexpressing S2 cells compared with the expression in the controls, whereas the levels of Profilin are reduced and the levels of Rho1 are unchanged. Error bars represent the S.E.M of at least 3 independent experiments; NS, not significant; *P

    Journal: Cell Communication and Signaling : CCS

    Article Title: Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila

    doi: 10.1186/s12964-018-0305-3

    Figure Lengend Snippet: Overexpression of jumu induces cell spreading and large numbers of filopodia in hemocytes. a-d Phalloidin staining (green) shows that Hml > GFP > UAS-jumu displays an increased number of filopodia and larger lamellipodia than the control ( a and b ); knockdown of ena or fascin in Hml > GFP > UAS-jumu can inhibit cell spreading and the formation of numerous filopodia ( c and d ). e-g Quantification of filopodium numbers and lengths and lamellipodial area. h Quantification of the phagocytosis indexes for latex beads, B. bassiana , S. aureus or E. coli. i-l Quantification of Ena, Fascin, Rho1 and Profilin levels. m Western blot analysis using antibodies against Ena, Fascin, Profilin and Rho1 and the corresponding graphical representation show that the levels of Ena and Fascin are increased in jumu -overexpressing S2 cells compared with the expression in the controls, whereas the levels of Profilin are reduced and the levels of Rho1 are unchanged. Error bars represent the S.E.M of at least 3 independent experiments; NS, not significant; *P

    Article Snippet: Then, 30 μg of the lysate was loaded into a 12% SDS-PAGE gel, followed by electroblotting onto nitrocellulose membranes and probing with mouse anti-α-tubulin (1:500, Sigma), mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (1:300, Developmental Studies Hybridoma Bank) for 2 h. The blot was subsequently probed with anti-mouse HRP-conjugated secondary antibodies for 1.5 h and detected using the ECL Plus detection system (Program).

    Techniques: Over Expression, Staining, Western Blot, Expressing

    Expression levels of proteins associated with actin filopodium formation are changed in jumu knockdown hemocytes. a Immunostaining against Ena (red) and Fascin (red) shows that the expression of Ena and Fascin is reduced in jumu knockdown hemocytes compared with the expression in the controls; however, the knockdown of NimC1 does not affect the expression of Ena and Fascin. b, c Quantification of signal intensities. d Real-time PCR analysis of ena , fascin , profilin and rho1 levels in jumu knockdown hemocytes. e-g Phalloidin staining (green) shows that the number and length of filopodia are reduced in the circulating hemocytes of Hml > GFP > ena RNAi and Hml > GFP > sn RNAi . h-j Circulating hemocytes of Hml > GFP > ena RNAi and Hml > GFP > sn RNAi isolated from third-instar larvae injected with latex beads (red) 1 h postinjection show defects in filopodia (green). k, l Quantification of the percentage of engulfing cells and phagocytic indexes based on phagocytosis assays. m-o’ Immunostaining against Ena (red) and phalloidin staining (green) shows that Ena is enriched at the tips of filopodia and lamellipodia in control circulating hemocytes; however, the expression level of Ena is markedly reduced at the tips of filopodia and lamellipodia in Hml > GFP > jumu RNAi (n and n’) and Hml > GFP > NimC1 RNAi (o and o’) circulating hemocytes. Error bars represent the S.E.M of at least 3 independent experiments; NS, not significant; *P

    Journal: Cell Communication and Signaling : CCS

    Article Title: Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila

    doi: 10.1186/s12964-018-0305-3

    Figure Lengend Snippet: Expression levels of proteins associated with actin filopodium formation are changed in jumu knockdown hemocytes. a Immunostaining against Ena (red) and Fascin (red) shows that the expression of Ena and Fascin is reduced in jumu knockdown hemocytes compared with the expression in the controls; however, the knockdown of NimC1 does not affect the expression of Ena and Fascin. b, c Quantification of signal intensities. d Real-time PCR analysis of ena , fascin , profilin and rho1 levels in jumu knockdown hemocytes. e-g Phalloidin staining (green) shows that the number and length of filopodia are reduced in the circulating hemocytes of Hml > GFP > ena RNAi and Hml > GFP > sn RNAi . h-j Circulating hemocytes of Hml > GFP > ena RNAi and Hml > GFP > sn RNAi isolated from third-instar larvae injected with latex beads (red) 1 h postinjection show defects in filopodia (green). k, l Quantification of the percentage of engulfing cells and phagocytic indexes based on phagocytosis assays. m-o’ Immunostaining against Ena (red) and phalloidin staining (green) shows that Ena is enriched at the tips of filopodia and lamellipodia in control circulating hemocytes; however, the expression level of Ena is markedly reduced at the tips of filopodia and lamellipodia in Hml > GFP > jumu RNAi (n and n’) and Hml > GFP > NimC1 RNAi (o and o’) circulating hemocytes. Error bars represent the S.E.M of at least 3 independent experiments; NS, not significant; *P

    Article Snippet: Then, 30 μg of the lysate was loaded into a 12% SDS-PAGE gel, followed by electroblotting onto nitrocellulose membranes and probing with mouse anti-α-tubulin (1:500, Sigma), mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (1:300, Developmental Studies Hybridoma Bank) for 2 h. The blot was subsequently probed with anti-mouse HRP-conjugated secondary antibodies for 1.5 h and detected using the ECL Plus detection system (Program).

    Techniques: Expressing, Immunostaining, Real-time Polymerase Chain Reaction, Staining, Isolation, Injection

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    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: Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).

    Techniques: Inhibition, Staining, Binding Assay

    Immunofluorescence double-staining to phenotype the P. acnes -positive cells in non-granulomatous areas of sarcoid lungs and lymph nodes. Green signal (FITC): PAB antibody; red signals (TRITC): anti-CD68 antibody ( a , c , e ), anti-fascin antibody ( b , d , f ). Results of double-staining are merged in all pictures. In the sarcoid lung, all of the P. acnes signals overlapped (yellow) with CD68-positive alveolar macrophages ( a ), whereas the fascin-positive alveolar dendritic cells did not contain small round bodies detected by PAB antibody ( b ). In the paracortical area of the sarcoid lymph node, small round body detected by PAB antibody were found in the CD68-positive macrophages ( c ), but not in the fascin-positive dendritic cells ( d ). In the sinus of the sarcoid lymph node, Hamazaki-Wesenberg bodies detected by PAB antibody overlapped with CD68-positive macrophages ( e ), but not with the fascin-positive dendritic cells ( f ). Original magnification: a – b , × 400; c – f , × 1000.

    Journal: Modern Pathology

    Article Title: Localization of Propionibacterium acnes in granulomas supports a possible etiologic link between sarcoidosis and the bacterium

    doi: 10.1038/modpathol.2012.80

    Figure Lengend Snippet: Immunofluorescence double-staining to phenotype the P. acnes -positive cells in non-granulomatous areas of sarcoid lungs and lymph nodes. Green signal (FITC): PAB antibody; red signals (TRITC): anti-CD68 antibody ( a , c , e ), anti-fascin antibody ( b , d , f ). Results of double-staining are merged in all pictures. In the sarcoid lung, all of the P. acnes signals overlapped (yellow) with CD68-positive alveolar macrophages ( a ), whereas the fascin-positive alveolar dendritic cells did not contain small round bodies detected by PAB antibody ( b ). In the paracortical area of the sarcoid lymph node, small round body detected by PAB antibody were found in the CD68-positive macrophages ( c ), but not in the fascin-positive dendritic cells ( d ). In the sinus of the sarcoid lymph node, Hamazaki-Wesenberg bodies detected by PAB antibody overlapped with CD68-positive macrophages ( e ), but not with the fascin-positive dendritic cells ( f ). Original magnification: a – b , × 400; c – f , × 1000.

    Article Snippet: Immunofluorescence Double-Staining According to the methods described previously, formalin-fixed and paraffin-embedded tissue sections of sarcoid lymph nodes with many P. acnes -positive cells were used for immunofluorescence double-staining to phenotype the cells with intracellular P. acnes using the antibodies to phagocytes, anti-human CD68 monoclonal antibody (clone KP1; DAKO) for macrophages, and anti-human fascin monoclonal antibody (clone 55K-2; DAKO) for dendritic cells.

    Techniques: Immunofluorescence, Double Staining

    RSK2 activates CREB to promote cancer cell invasion and migration by up-regulating proinvasive Fascin-1. A , stable knockdown of RSK2 or CREB by shRNA ( lower panels ) results in significantly decreased invasion ( upper panels ) in the metastatic cell line

    Journal: The Journal of Biological Chemistry

    Article Title: The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis *

    doi: 10.1074/jbc.M113.500561

    Figure Lengend Snippet: RSK2 activates CREB to promote cancer cell invasion and migration by up-regulating proinvasive Fascin-1. A , stable knockdown of RSK2 or CREB by shRNA ( lower panels ) results in significantly decreased invasion ( upper panels ) in the metastatic cell line

    Article Snippet: The image clone for Fascin-1 (GenBankTM accession no. ) was purchased from Open Biosystems.

    Techniques: Migration, shRNA

    RSK2 signals through CREB → Fascin-1 to promote filopodia formation in metastatic cancer cells. A549 and SKBR3 cells were seeded on silicon chips and evaluated with SEM. A , representative SEM images show the filopodia formed on the surface of

    Journal: The Journal of Biological Chemistry

    Article Title: The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis *

    doi: 10.1074/jbc.M113.500561

    Figure Lengend Snippet: RSK2 signals through CREB → Fascin-1 to promote filopodia formation in metastatic cancer cells. A549 and SKBR3 cells were seeded on silicon chips and evaluated with SEM. A , representative SEM images show the filopodia formed on the surface of

    Article Snippet: The image clone for Fascin-1 (GenBankTM accession no. ) was purchased from Open Biosystems.

    Techniques:

    RSK2 requires Fascin-1 to promote filopodia bundling. A , immunofluorescence staining results show the filopodia bundling detected by staining filopodia-associated actin filaments using Alexa Fluor 555-conjugated phalloidin in A549 cells. Stable RSK2 knockdown

    Journal: The Journal of Biological Chemistry

    Article Title: The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis *

    doi: 10.1074/jbc.M113.500561

    Figure Lengend Snippet: RSK2 requires Fascin-1 to promote filopodia bundling. A , immunofluorescence staining results show the filopodia bundling detected by staining filopodia-associated actin filaments using Alexa Fluor 555-conjugated phalloidin in A549 cells. Stable RSK2 knockdown

    Article Snippet: The image clone for Fascin-1 (GenBankTM accession no. ) was purchased from Open Biosystems.

    Techniques: Immunofluorescence, Staining

    The levels of RSK2, phospho-CREB, and Fascin-1 correlate with one another in primary human tumor tissue samples from HNSCC patients. A , IHC analysis was performed using 101 primary patient tissue specimens, including Tu (Met−) , Tu (Met+) , and LN

    Journal: The Journal of Biological Chemistry

    Article Title: The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis *

    doi: 10.1074/jbc.M113.500561

    Figure Lengend Snippet: The levels of RSK2, phospho-CREB, and Fascin-1 correlate with one another in primary human tumor tissue samples from HNSCC patients. A , IHC analysis was performed using 101 primary patient tissue specimens, including Tu (Met−) , Tu (Met+) , and LN

    Article Snippet: The image clone for Fascin-1 (GenBankTM accession no. ) was purchased from Open Biosystems.

    Techniques: Immunohistochemistry

    Treatment with RSK inhibitor FMK-MEA significantly attenuates activation of RSK2-CREB-Fascin-1 pathway in metastatic cells in vivo . A–C , IHC staining of phospho-RSK2 Ser-386 ( A ), phospho-CREB Ser-133 ( B ), and Fascin-1 ( C ) using LN samples from

    Journal: The Journal of Biological Chemistry

    Article Title: The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis *

    doi: 10.1074/jbc.M113.500561

    Figure Lengend Snippet: Treatment with RSK inhibitor FMK-MEA significantly attenuates activation of RSK2-CREB-Fascin-1 pathway in metastatic cells in vivo . A–C , IHC staining of phospho-RSK2 Ser-386 ( A ), phospho-CREB Ser-133 ( B ), and Fascin-1 ( C ) using LN samples from

    Article Snippet: The image clone for Fascin-1 (GenBankTM accession no. ) was purchased from Open Biosystems.

    Techniques: Microelectrode Array, Activation Assay, In Vivo, Immunohistochemistry, Staining

    RSK2-CREB pathway promotes cancer cell invasion and tumor metastasis in part by signaling through up-regulating Fascin-1. A , stable knockdown of Fascin-1 attenuates cell invasion of diverse metastatic cancer cells. B , expression of FLAG-tagged Fascin-1

    Journal: The Journal of Biological Chemistry

    Article Title: The Prometastatic Ribosomal S6 Kinase 2-cAMP Response Element-binding Protein (RSK2-CREB) Signaling Pathway Up-regulates the Actin-binding Protein Fascin-1 to Promote Tumor Metastasis *

    doi: 10.1074/jbc.M113.500561

    Figure Lengend Snippet: RSK2-CREB pathway promotes cancer cell invasion and tumor metastasis in part by signaling through up-regulating Fascin-1. A , stable knockdown of Fascin-1 attenuates cell invasion of diverse metastatic cancer cells. B , expression of FLAG-tagged Fascin-1

    Article Snippet: The image clone for Fascin-1 (GenBankTM accession no. ) was purchased from Open Biosystems.

    Techniques: Expressing

    Fascin is upregulated in migrating neuroblasts. A , Immunostaining of sagittal brain sections shows strong fascin expression along the RMS in both P7 and adult mice. B , C , Confocal images from P7 mouse SVZ sections showing that fascin immunostaining virtually overlaps with Dcx+ migrating neuroblasts ( B ), but is excluded from GFAP+ stem cells and astrocytes ( C ). D , Hardly any colocalization is observed with Mash1+ transit-amplifying progenitors. Scale bars: A , 500 μm; B–D , 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Fascin Regulates the Migration of Subventricular Zone-Derived Neuroblasts in the Postnatal Brain

    doi: 10.1523/JNEUROSCI.0653-13.2013

    Figure Lengend Snippet: Fascin is upregulated in migrating neuroblasts. A , Immunostaining of sagittal brain sections shows strong fascin expression along the RMS in both P7 and adult mice. B , C , Confocal images from P7 mouse SVZ sections showing that fascin immunostaining virtually overlaps with Dcx+ migrating neuroblasts ( B ), but is excluded from GFAP+ stem cells and astrocytes ( C ). D , Hardly any colocalization is observed with Mash1+ transit-amplifying progenitors. Scale bars: A , 500 μm; B–D , 10 μm.

    Article Snippet: Antibodies used were as follows: mouse anti-fascin and rabbit anti-GFAP (Dako); mouse anti-bromodeoxyuridine (BrdU) and rabbit anti-PKCα (BD Biosciences); rabbit anti-βIII-tubulin, anti-doublecortin (Dcx), and anti-Mash1 (Abcam); rabbit anti-GFP (Invitrogen); and rabbit anti-PKCγ (Santa Cruz Biotechnology).

    Techniques: Immunostaining, Expressing, Mouse Assay

    Genetic deletion of fascin affects neuroblast migration but not SVZ cell proliferation. A , To analyze cell proliferation, 2 h after injecting wt and fascin-1ko mice with BrdU coronal brain sections were prepared for staining with anti-BrdU antibodies. Representative pictures showing BrdU+ cells in the SVZ (brown), showing no major difference in the amount of BrdU+ cells between wt and fascin-1ko samples. LV, Lateral ventricle. B , To analyze SVZ-to-OB migration, P7 mice were injected with BrdU for 3 consecutive days. Twelve days later, sagittal brain slices were stained for Dcx and BrdU. Schematic diagram indicating the RMS/OB areas (areas 1 and 2) considered for quantification of BrdU+ cells. C , Representative images of BrdU+ cells (green) in areas 1 and 2 of the RMS in wt and fascin1-ko mice. Sections were also stained for Dcx-labeled migrating neuroblasts (red). D , Fascin-1ko animals display impaired migration, as shown by the increased percentage of BrdU+ cells in the caudal RMS and the decreased percentage of cells in the OB (mean ± SEM; n = 4 brains for wt mice; and n = 3 brains for fascin-1ko mice; * p

    Journal: The Journal of Neuroscience

    Article Title: Fascin Regulates the Migration of Subventricular Zone-Derived Neuroblasts in the Postnatal Brain

    doi: 10.1523/JNEUROSCI.0653-13.2013

    Figure Lengend Snippet: Genetic deletion of fascin affects neuroblast migration but not SVZ cell proliferation. A , To analyze cell proliferation, 2 h after injecting wt and fascin-1ko mice with BrdU coronal brain sections were prepared for staining with anti-BrdU antibodies. Representative pictures showing BrdU+ cells in the SVZ (brown), showing no major difference in the amount of BrdU+ cells between wt and fascin-1ko samples. LV, Lateral ventricle. B , To analyze SVZ-to-OB migration, P7 mice were injected with BrdU for 3 consecutive days. Twelve days later, sagittal brain slices were stained for Dcx and BrdU. Schematic diagram indicating the RMS/OB areas (areas 1 and 2) considered for quantification of BrdU+ cells. C , Representative images of BrdU+ cells (green) in areas 1 and 2 of the RMS in wt and fascin1-ko mice. Sections were also stained for Dcx-labeled migrating neuroblasts (red). D , Fascin-1ko animals display impaired migration, as shown by the increased percentage of BrdU+ cells in the caudal RMS and the decreased percentage of cells in the OB (mean ± SEM; n = 4 brains for wt mice; and n = 3 brains for fascin-1ko mice; * p

    Article Snippet: Antibodies used were as follows: mouse anti-fascin and rabbit anti-GFAP (Dako); mouse anti-bromodeoxyuridine (BrdU) and rabbit anti-PKCα (BD Biosciences); rabbit anti-βIII-tubulin, anti-doublecortin (Dcx), and anti-Mash1 (Abcam); rabbit anti-GFP (Invitrogen); and rabbit anti-PKCγ (Santa Cruz Biotechnology).

    Techniques: Migration, Mouse Assay, Staining, Injection, Labeling

    Fascin-1ko mice show abnormal neuroblast chain organization in the RMS. A , Immunostaining of the SVZ/RMS in sagittal brain slices from P7 mice showing the absence of fascin in fascin-1ko animals. B , Dcx-positive neuroblast chains appear thinner in fascin-1ko mice compared with wt mice. C , Lack of fascin does not appear to perturb the localization of GFAP+ astrocytes and stem cells. The dotted lines outline the RMS borders. Scale bars: A , B , 20 μm; C , 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Fascin Regulates the Migration of Subventricular Zone-Derived Neuroblasts in the Postnatal Brain

    doi: 10.1523/JNEUROSCI.0653-13.2013

    Figure Lengend Snippet: Fascin-1ko mice show abnormal neuroblast chain organization in the RMS. A , Immunostaining of the SVZ/RMS in sagittal brain slices from P7 mice showing the absence of fascin in fascin-1ko animals. B , Dcx-positive neuroblast chains appear thinner in fascin-1ko mice compared with wt mice. C , Lack of fascin does not appear to perturb the localization of GFAP+ astrocytes and stem cells. The dotted lines outline the RMS borders. Scale bars: A , B , 20 μm; C , 50 μm.

    Article Snippet: Antibodies used were as follows: mouse anti-fascin and rabbit anti-GFAP (Dako); mouse anti-bromodeoxyuridine (BrdU) and rabbit anti-PKCα (BD Biosciences); rabbit anti-βIII-tubulin, anti-doublecortin (Dcx), and anti-Mash1 (Abcam); rabbit anti-GFP (Invitrogen); and rabbit anti-PKCγ (Santa Cruz Biotechnology).

    Techniques: Mouse Assay, Immunostaining

    Immunohistochemical expression of fascin in Type B3 thymoma. Fascin + DCs (arrowheads) with the same strong intensity as endothelial cells (arrow) were scattered along with lymphocytic infiltration, while the tumor epithelium was weakly stained. Magnification,

    Journal: Oncology Letters

    Article Title: Fascin expression in dendritic cells and tumor epithelium in thymoma and thymic carcinoma

    doi: 10.3892/ol.2011.383

    Figure Lengend Snippet: Immunohistochemical expression of fascin in Type B3 thymoma. Fascin + DCs (arrowheads) with the same strong intensity as endothelial cells (arrow) were scattered along with lymphocytic infiltration, while the tumor epithelium was weakly stained. Magnification,

    Article Snippet: Additionally, the immunohistochemical staining of fascin was performed using a mouse monoclonal anti-fascin antibody (clone 55K-2, dilution 1:500; Dako, Glostrup, Denmark) on 4-μm paraffin-embedded sections, using a LSAB method by the Nex-ES IHC staining module (Ventana I-VIEW DAB universal kit; Ventana Medical Systems Inc., Tucson, AZ, USA).

    Techniques: Immunohistochemistry, Expressing, Staining

    No epithelial immunostaining for fascin was identified in thymic carcinoma, although endothelial cells and a small number of DCs were stained by fascin. Magnification, ×200.

    Journal: Oncology Letters

    Article Title: Fascin expression in dendritic cells and tumor epithelium in thymoma and thymic carcinoma

    doi: 10.3892/ol.2011.383

    Figure Lengend Snippet: No epithelial immunostaining for fascin was identified in thymic carcinoma, although endothelial cells and a small number of DCs were stained by fascin. Magnification, ×200.

    Article Snippet: Additionally, the immunohistochemical staining of fascin was performed using a mouse monoclonal anti-fascin antibody (clone 55K-2, dilution 1:500; Dako, Glostrup, Denmark) on 4-μm paraffin-embedded sections, using a LSAB method by the Nex-ES IHC staining module (Ventana I-VIEW DAB universal kit; Ventana Medical Systems Inc., Tucson, AZ, USA).

    Techniques: Immunostaining, Staining

    Borderline between Type A (upper right) and Type B areas (lower left) in Type AB thymoma (same fields in A-E). (A) Fascin immunostaining showed positive reactions for DCs and weak positive reactions for tumor epithelium. (B) HLA-DR immunostaining marked

    Journal: Oncology Letters

    Article Title: Fascin expression in dendritic cells and tumor epithelium in thymoma and thymic carcinoma

    doi: 10.3892/ol.2011.383

    Figure Lengend Snippet: Borderline between Type A (upper right) and Type B areas (lower left) in Type AB thymoma (same fields in A-E). (A) Fascin immunostaining showed positive reactions for DCs and weak positive reactions for tumor epithelium. (B) HLA-DR immunostaining marked

    Article Snippet: Additionally, the immunohistochemical staining of fascin was performed using a mouse monoclonal anti-fascin antibody (clone 55K-2, dilution 1:500; Dako, Glostrup, Denmark) on 4-μm paraffin-embedded sections, using a LSAB method by the Nex-ES IHC staining module (Ventana I-VIEW DAB universal kit; Ventana Medical Systems Inc., Tucson, AZ, USA).

    Techniques: Immunostaining

    Inhibition of actin-bundling using Fascin-specific nanobodies displays a significant reduction of virus transmission. (A-C) 293T cells were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmid pCMVHT1M-ΔEnv encoding all HTLV-1 proteins except env (Δenv) pseudotyped with VSV-G. Cells were co-transfected with V5-tagged expression plasmids encoding a mitochondrial outer membrane (MOM) sequence and nanobodies (Nb) targeting Fascin (MOMFASNb2 (FASNb2), 0.5μg; or MOMFASNb5 (FASNb5), 0.25; 0.5; 1μg), or a control nanobody (MOMGFPNb (GFPNb), 0.5μg). At 48h post transfection, luciferase assays, ELISA and western blot were performed as described in Fig 1A . The means of four independent experiments ± standard error (SE) are shown and were compared to the control (GFPNb) using Student’s t-test (**: p

    Journal: PLoS Pathogens

    Article Title: The Tax-Inducible Actin-Bundling Protein Fascin Is Crucial for Release and Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1 (HTLV-1)

    doi: 10.1371/journal.ppat.1005916

    Figure Lengend Snippet: Inhibition of actin-bundling using Fascin-specific nanobodies displays a significant reduction of virus transmission. (A-C) 293T cells were transfected with the reporter vector pCRU5HT1M-inluc (inluc) and the packaging plasmid pCMVHT1M-ΔEnv encoding all HTLV-1 proteins except env (Δenv) pseudotyped with VSV-G. Cells were co-transfected with V5-tagged expression plasmids encoding a mitochondrial outer membrane (MOM) sequence and nanobodies (Nb) targeting Fascin (MOMFASNb2 (FASNb2), 0.5μg; or MOMFASNb5 (FASNb5), 0.25; 0.5; 1μg), or a control nanobody (MOMGFPNb (GFPNb), 0.5μg). At 48h post transfection, luciferase assays, ELISA and western blot were performed as described in Fig 1A . The means of four independent experiments ± standard error (SE) are shown and were compared to the control (GFPNb) using Student’s t-test (**: p

    Article Snippet: After SDS-PAGE and immunoblotting on nitrocellulose transfer membranes (Whatmann, Protran, Whatmann GmbH, Dassel, Germany), proteins were detected using the following antibodies: rabbit polyclonal antibodies anti-V5 (Sigma), mouse monoclonal antibodies anti-Fascin (55K-2; Dako Deutschland GmbH, Hamburg, Germany), anti-β-actin (ACTB; Sigma), anti-Hsp90 α/β (F-8; Santa Cruz Biotechnology, Heidelberg, Germany), anti-HTLV-1 gag p19 (ZeptoMetrix Corporation), and anti-GFP (Sigma), and mouse antibodies to Tax, which were derived from the hybridoma cell line 168B17-46-34 (provided by B. Langton through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH; [ ]).

    Techniques: Inhibition, Transmission Assay, Transfection, Plasmid Preparation, Expressing, Sequencing, Luciferase, Enzyme-linked Immunosorbent Assay, Western Blot

    Fascin and gag localize at cell-cell contacts and in long-distance connections between infected and uninfected T-cells. (A) Confocal laser scanning microscopy of HTLV-1-infected MS-9 cells co-cultured with Jurkat T-cells. Jurkat T-cells were pre-stained with Calcein-AM (green) to differentiate between the two cell types. Cells were co-cultured for 0 (k-p), 30 (a-j) or 60min (q-w) on poly-L-lysine-coated coverslips prior to drying (20min), fixation, and staining. (A) Stainings of Calcein (green), gag (blue), Fascin (red) and the merge of all three stainings are shown. Transmitted light served as control. Representative stainings of three independent experiments showing clusters of Fascin (a-e) and gag (a-j), Fascin clutches (f-j) or long-distance connections (k-w) are depicted. Thin white arrows indicate gag of an infected cell clustering at the cell-cell contact towards an uninfected cell; framed white arrows indicate short-distance Fascin-containing membrane extensions; and thick white arrows indicate long-distance protrusions between uninfected and infected cells. Protrusions (k-o; q-u) were examined in more detail, and (p) the stains of gag and Fascin within the protrusion shown in (n) were enlarged; further, a region of interest (v-w) was analyzed showing the intensities of gag- (blue) and Fascin- specific (red) fluorescences shown in (t). (B) Detection of Fascin, Tax-1 and gag in HTLV-1-infected MS-9 cells and uninfected Jurkat T-cells by western blot. Hsp90 α/β served as control.

    Journal: PLoS Pathogens

    Article Title: The Tax-Inducible Actin-Bundling Protein Fascin Is Crucial for Release and Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1 (HTLV-1)

    doi: 10.1371/journal.ppat.1005916

    Figure Lengend Snippet: Fascin and gag localize at cell-cell contacts and in long-distance connections between infected and uninfected T-cells. (A) Confocal laser scanning microscopy of HTLV-1-infected MS-9 cells co-cultured with Jurkat T-cells. Jurkat T-cells were pre-stained with Calcein-AM (green) to differentiate between the two cell types. Cells were co-cultured for 0 (k-p), 30 (a-j) or 60min (q-w) on poly-L-lysine-coated coverslips prior to drying (20min), fixation, and staining. (A) Stainings of Calcein (green), gag (blue), Fascin (red) and the merge of all three stainings are shown. Transmitted light served as control. Representative stainings of three independent experiments showing clusters of Fascin (a-e) and gag (a-j), Fascin clutches (f-j) or long-distance connections (k-w) are depicted. Thin white arrows indicate gag of an infected cell clustering at the cell-cell contact towards an uninfected cell; framed white arrows indicate short-distance Fascin-containing membrane extensions; and thick white arrows indicate long-distance protrusions between uninfected and infected cells. Protrusions (k-o; q-u) were examined in more detail, and (p) the stains of gag and Fascin within the protrusion shown in (n) were enlarged; further, a region of interest (v-w) was analyzed showing the intensities of gag- (blue) and Fascin- specific (red) fluorescences shown in (t). (B) Detection of Fascin, Tax-1 and gag in HTLV-1-infected MS-9 cells and uninfected Jurkat T-cells by western blot. Hsp90 α/β served as control.

    Article Snippet: After SDS-PAGE and immunoblotting on nitrocellulose transfer membranes (Whatmann, Protran, Whatmann GmbH, Dassel, Germany), proteins were detected using the following antibodies: rabbit polyclonal antibodies anti-V5 (Sigma), mouse monoclonal antibodies anti-Fascin (55K-2; Dako Deutschland GmbH, Hamburg, Germany), anti-β-actin (ACTB; Sigma), anti-Hsp90 α/β (F-8; Santa Cruz Biotechnology, Heidelberg, Germany), anti-HTLV-1 gag p19 (ZeptoMetrix Corporation), and anti-GFP (Sigma), and mouse antibodies to Tax, which were derived from the hybridoma cell line 168B17-46-34 (provided by B. Langton through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH; [ ]).

    Techniques: Infection, Confocal Laser Scanning Microscopy, Mass Spectrometry, Cell Culture, Staining, Western Blot

    Resistin expression was associated with the survival of patients with colorectal cancer. (a, b). The associations of resistin expression with relapse-free survival (RFS) (a) and overall survival (OS) (b) were analyzed. (c, d). The associations of fascin-1 expression with RFS (c) and OS (d) were analyzed. (e, f). Kaplan-Meier curves for OS (e) and RFS (f) of combined high expression of resistin and fascin-1 in colorectal cancer. P values were calculated using the Mantel-Cox log-rank test.

    Journal: BioMed Research International

    Article Title: High Expression of Both Resistin and Fascin-1 Predicts a Poor Prognosis in Patients with Colorectal Cancer

    doi: 10.1155/2020/8753175

    Figure Lengend Snippet: Resistin expression was associated with the survival of patients with colorectal cancer. (a, b). The associations of resistin expression with relapse-free survival (RFS) (a) and overall survival (OS) (b) were analyzed. (c, d). The associations of fascin-1 expression with RFS (c) and OS (d) were analyzed. (e, f). Kaplan-Meier curves for OS (e) and RFS (f) of combined high expression of resistin and fascin-1 in colorectal cancer. P values were calculated using the Mantel-Cox log-rank test.

    Article Snippet: The primary antibodies used included anti-resistin mouse monoclonal antibody (clone C-10, diluted at 1 : 25; Santa Cruz Biotechnology, Santa Cruz, USA) and anti-fascin-1 mouse monoclonal antibody (clone 55k-2, diluted at 1 : 100; Santa Cruz Biotechnology).

    Techniques: Expressing

    A tendency of positive protein levels between resistin and fascin-1 in colorectal cancer: human colorectal cancer tissue microarrays were immune-stained with anti-resistin and anti-fascin-1 antibodies. Representative staining pictures of tumors are shown.

    Journal: BioMed Research International

    Article Title: High Expression of Both Resistin and Fascin-1 Predicts a Poor Prognosis in Patients with Colorectal Cancer

    doi: 10.1155/2020/8753175

    Figure Lengend Snippet: A tendency of positive protein levels between resistin and fascin-1 in colorectal cancer: human colorectal cancer tissue microarrays were immune-stained with anti-resistin and anti-fascin-1 antibodies. Representative staining pictures of tumors are shown.

    Article Snippet: The primary antibodies used included anti-resistin mouse monoclonal antibody (clone C-10, diluted at 1 : 25; Santa Cruz Biotechnology, Santa Cruz, USA) and anti-fascin-1 mouse monoclonal antibody (clone 55k-2, diluted at 1 : 100; Santa Cruz Biotechnology).

    Techniques: Staining

    TnTs express proteins characteristic of actin-based extensions. Confocal imaging demonstrated immunofluorescence of specific protein components of MSTO-211H cells with stained TnTs. a) Actin is uniform across the entirety of the TnT, which passes over an adherent cell. b) Fascin is expressed intermittently and at the base of nanotubes c) Pankeratin localizes to the perinuclear region of the cells. d) Ezrin expression was most prominent at the base of TnTs, consistent with its role in organizing actin-based filaments. e) ß-catenin is minimally present within TnTs. f) E-cadherin staining of TnTs between cells. g) ZO-1 localizes to the cell membrane, including the point of contact of TnTs. Scale bars: a) 20 µm, b) 20 µm, c) 30 µm, d) 30 µm, e) 20 µm, f) 20 µm, g) 50 µm.

    Journal: PLoS ONE

    Article Title: Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma

    doi: 10.1371/journal.pone.0033093

    Figure Lengend Snippet: TnTs express proteins characteristic of actin-based extensions. Confocal imaging demonstrated immunofluorescence of specific protein components of MSTO-211H cells with stained TnTs. a) Actin is uniform across the entirety of the TnT, which passes over an adherent cell. b) Fascin is expressed intermittently and at the base of nanotubes c) Pankeratin localizes to the perinuclear region of the cells. d) Ezrin expression was most prominent at the base of TnTs, consistent with its role in organizing actin-based filaments. e) ß-catenin is minimally present within TnTs. f) E-cadherin staining of TnTs between cells. g) ZO-1 localizes to the cell membrane, including the point of contact of TnTs. Scale bars: a) 20 µm, b) 20 µm, c) 30 µm, d) 30 µm, e) 20 µm, f) 20 µm, g) 50 µm.

    Article Snippet: Immunofluorescent Staining The primary antibodies and their working concentrations are as follows: rabbit anti-β-catenin (Sigma, 5 µg/mL), mouse anti-Ezrin (Santa Cruz, 5 µg/mL), mouse anti-E-Cadherin (BD Bioscience, 2.5 µg/mL), mouse anti-human Fascin (Dako, 1 µg/mL), mouse anti-ZO1 (Zymed, 1 µg/mL), mouse anti-GM130 (BD Transduction, 1 µg/mL).

    Techniques: Imaging, Immunofluorescence, Staining, Expressing

    Validation and characterization of OS cell lines with altered Fascin-1 expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a pLenti6/V5-DEST empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)

    Journal: BMC Cancer

    Article Title: Fascin-1 enhances experimental osteosarcoma tumor formation and metastasis and is related to poor patient outcome

    doi: 10.1186/s12885-019-5303-3

    Figure Lengend Snippet: Validation and characterization of OS cell lines with altered Fascin-1 expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a pLenti6/V5-DEST empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)

    Article Snippet: SaOS-2-LacZ and 143B-LacZ cells stably over-expressing Fascin-1 (SaOS-2/Fascin-1 and 143B/Fascin-1) were then obtained by transduction with pLenti6/V5-DEST-FASCIN (a gift from Lynda Chin; Addgene plasmid # 31207) and subsequent selection with Blasticidin (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Western Blot, Stable Transfection, Transduction, shRNA, Plasmid Preparation, Staining, Over Expression