fibronectin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher fibronectin
    ECM dependent morphological differences modulated through CD47 signalling. (A) Fluorescent microscope images were taken of cells plated onto cover slips coated either with 6% BSA, 10 µg/ml collagen I or <t>fibronectin</t> for 2 hours. Where indicated cells were pre-treated with 500 ng/ml PTX for 2 hours, 20 µg/ml of the CD47 functional blocking antibody for 20 minutes or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes. Cells were fixed, permeabilised and stained with primary antibodies against either CD47 or α2 integrin using either Alexa-488 or -546 conjugated secondary antibodies. (B) as in A but stained with primary antibodies against CD47, α2 integrin and F-actin. (C) as in A but stained with primary antibodies against Rho A and F-actin. Shown are representative pictures of three separate experiments taken at 60× magnification. (D) A graphical representation of the number of cells with membrane blebs from 100 cell samples. The statistical analyses were performed with unpaired Students t-test; **P
    Fibronectin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "CD47 Regulates Collagen I-Induced Cyclooxygenase-2 Expression and Intestinal Epithelial Cell Migration"

    Article Title: CD47 Regulates Collagen I-Induced Cyclooxygenase-2 Expression and Intestinal Epithelial Cell Migration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006371

    ECM dependent morphological differences modulated through CD47 signalling. (A) Fluorescent microscope images were taken of cells plated onto cover slips coated either with 6% BSA, 10 µg/ml collagen I or fibronectin for 2 hours. Where indicated cells were pre-treated with 500 ng/ml PTX for 2 hours, 20 µg/ml of the CD47 functional blocking antibody for 20 minutes or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes. Cells were fixed, permeabilised and stained with primary antibodies against either CD47 or α2 integrin using either Alexa-488 or -546 conjugated secondary antibodies. (B) as in A but stained with primary antibodies against CD47, α2 integrin and F-actin. (C) as in A but stained with primary antibodies against Rho A and F-actin. Shown are representative pictures of three separate experiments taken at 60× magnification. (D) A graphical representation of the number of cells with membrane blebs from 100 cell samples. The statistical analyses were performed with unpaired Students t-test; **P
    Figure Legend Snippet: ECM dependent morphological differences modulated through CD47 signalling. (A) Fluorescent microscope images were taken of cells plated onto cover slips coated either with 6% BSA, 10 µg/ml collagen I or fibronectin for 2 hours. Where indicated cells were pre-treated with 500 ng/ml PTX for 2 hours, 20 µg/ml of the CD47 functional blocking antibody for 20 minutes or 100 µM of the COX-2 specific inhibitor NS-398 for 30 minutes. Cells were fixed, permeabilised and stained with primary antibodies against either CD47 or α2 integrin using either Alexa-488 or -546 conjugated secondary antibodies. (B) as in A but stained with primary antibodies against CD47, α2 integrin and F-actin. (C) as in A but stained with primary antibodies against Rho A and F-actin. Shown are representative pictures of three separate experiments taken at 60× magnification. (D) A graphical representation of the number of cells with membrane blebs from 100 cell samples. The statistical analyses were performed with unpaired Students t-test; **P

    Techniques Used: Microscopy, Functional Assay, Blocking Assay, Staining

    2) Product Images from "Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix"

    Article Title: Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2016.08.011

    Effects of ECM dimensionality and architecture on neurites. (A) Neurites extended on decellularized ECM were stained with an anti-α-tubulin monoclonal antibody followed by fluorescein-goat anti-mouse IgG. The ECM was stained with R457 anti-fibronectin antiserum and rhodamine-goat anti-rabbit IgG. A merged image of confocal z-sections from the periphery of the explant is shown. Scale bar is 50 microns. (B) A perspective 3D view of the confocal z-stack for the image in A is shown. Sample is ∼ 12 microns thick. (C) The perspective 3D view from B was depth-coded for the neurites only. Colors represent different levels of depth within the sample with those regions of neurites that are the highest in blue (0 microns) and the yellow regions indicating neurites that are within the ECM (7-8 microns from the top).
    Figure Legend Snippet: Effects of ECM dimensionality and architecture on neurites. (A) Neurites extended on decellularized ECM were stained with an anti-α-tubulin monoclonal antibody followed by fluorescein-goat anti-mouse IgG. The ECM was stained with R457 anti-fibronectin antiserum and rhodamine-goat anti-rabbit IgG. A merged image of confocal z-sections from the periphery of the explant is shown. Scale bar is 50 microns. (B) A perspective 3D view of the confocal z-stack for the image in A is shown. Sample is ∼ 12 microns thick. (C) The perspective 3D view from B was depth-coded for the neurites only. Colors represent different levels of depth within the sample with those regions of neurites that are the highest in blue (0 microns) and the yellow regions indicating neurites that are within the ECM (7-8 microns from the top).

    Techniques Used: Staining

    3) Product Images from "Matrix Stiffness Modulates Mesenchymal Stem Cell Sensitivity to Geometric Asymmetry Signals"

    Article Title: Matrix Stiffness Modulates Mesenchymal Stem Cell Sensitivity to Geometric Asymmetry Signals

    Journal: Annals of biomedical engineering

    doi: 10.1007/s10439-018-2008-8

    Schematic of the patterning process. (A) UV lithography was used to create consistent patterns in the shape of the letters O, Y, and T. (B) Thiol groups allow selective binding of the adhesive fibronectin protein to patterned areas, as confirmed using fluorescent bovine serum albumin (C). Individual hMSCs adhered and conformed to the patterned regions. Scale is 100 μm.
    Figure Legend Snippet: Schematic of the patterning process. (A) UV lithography was used to create consistent patterns in the shape of the letters O, Y, and T. (B) Thiol groups allow selective binding of the adhesive fibronectin protein to patterned areas, as confirmed using fluorescent bovine serum albumin (C). Individual hMSCs adhered and conformed to the patterned regions. Scale is 100 μm.

    Techniques Used: Binding Assay

    4) Product Images from "Silk fibroin scaffolds with muscle‐like elasticity support in vitro differentiation of human skeletal muscle cells) Silk fibroin scaffolds with muscle‐like elasticity support in vitro differentiation of human skeletal muscle cells"

    Article Title: Silk fibroin scaffolds with muscle‐like elasticity support in vitro differentiation of human skeletal muscle cells) Silk fibroin scaffolds with muscle‐like elasticity support in vitro differentiation of human skeletal muscle cells

    Journal: Journal of Tissue Engineering and Regenerative Medicine

    doi: 10.1002/term.2227

    Human skeletal muscle myoblasts (HSMMs) are viable on silk fibroin substrates. Metabolic activity of HSMMs on four different silk fibroins, two extracellular matrix proteins and tissue culture plastic in Skeletal Muscle Growth Medium‐2 was assessed using the Alamar Blue assay. The days in culture when the Alamar Blue dye was added and absorbance measured are indicated. The relative fluorescence units (RFU) were calculated relative to the control (media, no cells). Data are mean ± standard deviation ( n = 4). Am, Antheraea mylitta ; Bm, Bombyx mori ; Aa, Antheraea assamensis ; Col I, collagen I; Fibro, Fibronectin; Sr, Samia ricini ; * p ≤ 0.05
    Figure Legend Snippet: Human skeletal muscle myoblasts (HSMMs) are viable on silk fibroin substrates. Metabolic activity of HSMMs on four different silk fibroins, two extracellular matrix proteins and tissue culture plastic in Skeletal Muscle Growth Medium‐2 was assessed using the Alamar Blue assay. The days in culture when the Alamar Blue dye was added and absorbance measured are indicated. The relative fluorescence units (RFU) were calculated relative to the control (media, no cells). Data are mean ± standard deviation ( n = 4). Am, Antheraea mylitta ; Bm, Bombyx mori ; Aa, Antheraea assamensis ; Col I, collagen I; Fibro, Fibronectin; Sr, Samia ricini ; * p ≤ 0.05

    Techniques Used: Activity Assay, Alamar Blue Assay, Fluorescence, Standard Deviation

    Human skeletal muscle myoblasts (HSMMs) adhere and secrete extracellular matrix (ECM) proteins on three‐dimensional (3D) silk scaffolds. The HSMMs were cultured on 3D silk scaffolds for 2 days in skeletal muscle growth medium‐2 proliferation medium, then fixed and stained with rhodamine–phalloidin (F‐actin staining; b,f,j,n), or antibodies recognizing either fibronectin (c,g,k,o), or perlecan (d,h,l,p). 4′,6‐diamidino‐2‐phenylindole (DAPI) stained the silk scaffolds (all panels). Images were captured using a Nikon A1 confocal laser scanning microscope. The merged images of several z‐stack images (5 μm each stack, 150–200 μm deep into the scaffold) are presented. A representative image of six fields of view is shown. Bar: 50 μm. [Colour figure can be viewed at wileyonlinelibrary.com ]
    Figure Legend Snippet: Human skeletal muscle myoblasts (HSMMs) adhere and secrete extracellular matrix (ECM) proteins on three‐dimensional (3D) silk scaffolds. The HSMMs were cultured on 3D silk scaffolds for 2 days in skeletal muscle growth medium‐2 proliferation medium, then fixed and stained with rhodamine–phalloidin (F‐actin staining; b,f,j,n), or antibodies recognizing either fibronectin (c,g,k,o), or perlecan (d,h,l,p). 4′,6‐diamidino‐2‐phenylindole (DAPI) stained the silk scaffolds (all panels). Images were captured using a Nikon A1 confocal laser scanning microscope. The merged images of several z‐stack images (5 μm each stack, 150–200 μm deep into the scaffold) are presented. A representative image of six fields of view is shown. Bar: 50 μm. [Colour figure can be viewed at wileyonlinelibrary.com ]

    Techniques Used: Cell Culture, Staining, Laser-Scanning Microscopy

    5) Product Images from "Deconstructing the Effects of Matrix Elasticity and Geometry in Mesenchymal Stem Cell Lineage Commitment"

    Article Title: Deconstructing the Effects of Matrix Elasticity and Geometry in Mesenchymal Stem Cell Lineage Commitment

    Journal: Advanced functional materials

    doi: 10.1002/adfm.201303400

    Schematic showing UV lithography process used to create hydrogel shapes of varying elasticity. Hydrogel shapes were functionalized with thiol to promote fibronectin binding exclusively to hydrogels.
    Figure Legend Snippet: Schematic showing UV lithography process used to create hydrogel shapes of varying elasticity. Hydrogel shapes were functionalized with thiol to promote fibronectin binding exclusively to hydrogels.

    Techniques Used: Binding Assay

    6) Product Images from "Resistin-like molecule-β (RELM-β) targets airways fibroblasts to effect remodelling in asthma: from mouse to man"

    Article Title: Resistin-like molecule-β (RELM-β) targets airways fibroblasts to effect remodelling in asthma: from mouse to man

    Journal: Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology

    doi: 10.1111/cea.12481

    (a) Photomicrographs showing immunoreactivity for αsmooth muscle actin, fibronectin and collagen I in bronchial biopsy sections from an asthmatic and a control subject. (b, d and f) Global immunoreactivity for α-SMA, fibronectin and collagen
    Figure Legend Snippet: (a) Photomicrographs showing immunoreactivity for αsmooth muscle actin, fibronectin and collagen I in bronchial biopsy sections from an asthmatic and a control subject. (b, d and f) Global immunoreactivity for α-SMA, fibronectin and collagen

    Techniques Used:

    7) Product Images from "THE FATE OF INTERNALIZED α5 INTEGRIN IS REGULATED BY MATRIX-CAPABLE FIBRONECTIN"

    Article Title: THE FATE OF INTERNALIZED α5 INTEGRIN IS REGULATED BY MATRIX-CAPABLE FIBRONECTIN

    Journal: The Journal of surgical research

    doi: 10.1016/j.jss.2014.05.084

    Degradation of internalized α5 integrin is MG-132 sensitive and in the absence of fibronectin, α5 integrin cytoplasmic tail is ubiquitinated and CBL associates with α5 integrin
    Figure Legend Snippet: Degradation of internalized α5 integrin is MG-132 sensitive and in the absence of fibronectin, α5 integrin cytoplasmic tail is ubiquitinated and CBL associates with α5 integrin

    Techniques Used:

    α5 integrin levels decrease in the absence of fibronectin and stabilization of internalized α5 requires ‘matrix-capable’ fibronectin
    Figure Legend Snippet: α5 integrin levels decrease in the absence of fibronectin and stabilization of internalized α5 requires ‘matrix-capable’ fibronectin

    Techniques Used:

    α5 integrin degradation in the absence of fibronectin requires the cytoplasmic tail of α5 integrin distal to the conserved GFFKR region and containing two lysine residues that are ubiquitinated
    Figure Legend Snippet: α5 integrin degradation in the absence of fibronectin requires the cytoplasmic tail of α5 integrin distal to the conserved GFFKR region and containing two lysine residues that are ubiquitinated

    Techniques Used:

    Loss of two carboxyl-terminal lysine residues results in a diminished rate of α5 integrin internalization and fibronectin matrix assembly, which is regulated by endocytosis
    Figure Legend Snippet: Loss of two carboxyl-terminal lysine residues results in a diminished rate of α5 integrin internalization and fibronectin matrix assembly, which is regulated by endocytosis

    Techniques Used:

    Fibronectin's impact on regulation of cell-surface α5 integrin
    Figure Legend Snippet: Fibronectin's impact on regulation of cell-surface α5 integrin

    Techniques Used:

    8) Product Images from "Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior"

    Article Title: Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037904

    Macromolecular crowding induces alignment of extracellular matrix fibers and an increase in deposition of collagen type-I in human bone marrow-derived mesenchymal stromal or stem cells (MSCs). ( A ) Atomic force microscopy (AFM) contact mode deflection images of extracellular matrix deposited by MSCs left behind after detergent removal of cells in media containing macromolecular crowders (+MMC media) and ( B ) −MMC media after 7 days. Scale bars = 2 mm. ( C ) Average angular standard deviation for AFM imaging of extracellular matrix in A and B (N = 5 +MMC, N = 5 −MMC, p = 0.1363). ( D ) Immunostaining of extracellular fibronectin (red) and cell nucleus (blue, DAPI) for +MMC and ( E ) −MMC in MSCs after 3 days of culture. Scale bars = 30 µm. ( F ) Average angular standard deviation for fibronectin in D and E (N = 15 +MMC, N = 11 −MMC, p = 0.0013). ( G ) Immunostaining of extracellular collagen IV (green) and cell nucleus (blue, DAPI) +MMC, ( H ) and −MMC, in hMSCs after 3 days of culture. Scale bars = 15 mm ( I ) Average angular standard deviation for collagen IV in G and H (N = 12 +MMC, N = 8 −MMC, p = 0.0253). Lower values of average angular standard deviation indicate a higher degree of alignment of fibers comprising the extracellular matrix. ( J ) Western blot of collagen type-I secreted by human bone marrow-derived mesenchymal stromal or stem cells after 48 hrs in culture medium ± MMC demonstrates a significant increase in the cell deposited collagen into the matrix +MMC. ( K ) Normalized densitometry of collagen type-I Western blot demonstrates a shift in collagen distribution from media to matrix, indicative of the enhanced deposition of collagen in the presence of MMCs. Significant increases in crosslinked collagen α-chains (β-bands) is observed in the cell deposited matrix +MMC, as compared to −MMC conditions (p
    Figure Legend Snippet: Macromolecular crowding induces alignment of extracellular matrix fibers and an increase in deposition of collagen type-I in human bone marrow-derived mesenchymal stromal or stem cells (MSCs). ( A ) Atomic force microscopy (AFM) contact mode deflection images of extracellular matrix deposited by MSCs left behind after detergent removal of cells in media containing macromolecular crowders (+MMC media) and ( B ) −MMC media after 7 days. Scale bars = 2 mm. ( C ) Average angular standard deviation for AFM imaging of extracellular matrix in A and B (N = 5 +MMC, N = 5 −MMC, p = 0.1363). ( D ) Immunostaining of extracellular fibronectin (red) and cell nucleus (blue, DAPI) for +MMC and ( E ) −MMC in MSCs after 3 days of culture. Scale bars = 30 µm. ( F ) Average angular standard deviation for fibronectin in D and E (N = 15 +MMC, N = 11 −MMC, p = 0.0013). ( G ) Immunostaining of extracellular collagen IV (green) and cell nucleus (blue, DAPI) +MMC, ( H ) and −MMC, in hMSCs after 3 days of culture. Scale bars = 15 mm ( I ) Average angular standard deviation for collagen IV in G and H (N = 12 +MMC, N = 8 −MMC, p = 0.0253). Lower values of average angular standard deviation indicate a higher degree of alignment of fibers comprising the extracellular matrix. ( J ) Western blot of collagen type-I secreted by human bone marrow-derived mesenchymal stromal or stem cells after 48 hrs in culture medium ± MMC demonstrates a significant increase in the cell deposited collagen into the matrix +MMC. ( K ) Normalized densitometry of collagen type-I Western blot demonstrates a shift in collagen distribution from media to matrix, indicative of the enhanced deposition of collagen in the presence of MMCs. Significant increases in crosslinked collagen α-chains (β-bands) is observed in the cell deposited matrix +MMC, as compared to −MMC conditions (p

    Techniques Used: Derivative Assay, Microscopy, Standard Deviation, Imaging, Immunostaining, Western Blot

    9) Product Images from "A micropatterning and image processing approach to simplify measurement of cellular traction forces"

    Article Title: A micropatterning and image processing approach to simplify measurement of cellular traction forces

    Journal: Acta Biomaterialia

    doi: 10.1016/j.actbio.2011.08.013

    A small drop of fluorescently labeled fibronectin was used to adsorb a circular region of fibronectin on a glass coverslip that was then transferred to the surface of a PAA gel containing NHS-ester during the polymerization phase. Fibroblast cells were
    Figure Legend Snippet: A small drop of fluorescently labeled fibronectin was used to adsorb a circular region of fibronectin on a glass coverslip that was then transferred to the surface of a PAA gel containing NHS-ester during the polymerization phase. Fibroblast cells were

    Techniques Used: Labeling

    A mold is created in PDMS that can stamp the desired pattern onto glass. Fluorescently labeled fibronectin in PBS is adsorbed onto the plasma-treated, hydrophilic stamp (A). After rinsing the surface, adsorbed fibronectin is then stamped onto the plasma-treated
    Figure Legend Snippet: A mold is created in PDMS that can stamp the desired pattern onto glass. Fluorescently labeled fibronectin in PBS is adsorbed onto the plasma-treated, hydrophilic stamp (A). After rinsing the surface, adsorbed fibronectin is then stamped onto the plasma-treated

    Techniques Used: Labeling

    A fluorescence image of the fluorescently labeled fibronectin pattern (A) is overlaid with the bright-field image of a fibroblast cell after 24 h in culture on the patterned PAA gel (B). First, the analysis program identifies the locations of the dots
    Figure Legend Snippet: A fluorescence image of the fluorescently labeled fibronectin pattern (A) is overlaid with the bright-field image of a fibroblast cell after 24 h in culture on the patterned PAA gel (B). First, the analysis program identifies the locations of the dots

    Techniques Used: Fluorescence, Labeling

    10) Product Images from "SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras"

    Article Title: SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

    Journal: Nature cell biology

    doi: 10.1038/ncb3487

    SHANK3 regulates integrin activation and cell spreading by a Rap1/Ras-dependent mechanism a , FACS analysis shows that SHANK3 WT-mRFP, but not SHANK3 mutant, overexpression prevents Rap1Q63E-mediated β1-integrin activation in HEK293 cells. Data represent mean ± SEM relative to mRFP and GFP expressing cells (n = 4 independent experiments; 5000 mRFP/GFP-positive cells analysed per experiment). b , c , Representative confocal images (b) and quantification (c) of spreading MDA-MB-231 cells transfected with GFP-Rap1Q63E alone or together with SHANK3 WT-mRFP. Staining: F-actin and DAPI (cell nuclei). Middle plane confocal image is shown. Scale bar: 10 μm. Tukey box plots represent median cell area relative to untransfected cells (n = 26, 21, 27 cells from left to right from three independent experiments). d,e , Representative confocal images (d) and quantification (e) of filopodia (indicated by arrowheads) in rat hippocampal neurons plated on laminin. Staining: F-actin and Map2 (neurite marker). Tukey box plots are shown (n = 176 (WT), 120 (L68P) from two independent experiments). Scale bar = 20 μm (original image) and 10 µm (ROI). f,g , Representative confocal images (f) and quantification (g) of filopodia in SK-N-BE-2 neuroblastoma cells plated on laminin and differentiated with retinoid acid (10 µM, three days). Shank3 SPN WT-GFP overexpression reduces filopodia density in neurites. Tukey box plots are shown (n = 76 (GFP), 62 (WT), three independent experiments). Scale bar = 20 μm. h , FACS analysis of β1-integrin activity in SHANK3- silenced HEK293 cells treated with a Rap1 inhibitor (10 μM, 1 h). (f) Data are mean ± SEM (n = 5 independent experiments; 10000 cells per experiment). i,j , Representative images (i) and Image J quantification (j) of cell area in SHANK3- silenced HEK293 cells ± Rap1 inhibitor (10 µM, 1 h). Cells were adhering on a fibronectin-collagen matrix (15 min). Middle plane confocal image is shown. Scale bar = 10 μm. Data are represented by Tukey box plots (n = 30, 33, 38 cells from left to right from two independent experiments. Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3 .
    Figure Legend Snippet: SHANK3 regulates integrin activation and cell spreading by a Rap1/Ras-dependent mechanism a , FACS analysis shows that SHANK3 WT-mRFP, but not SHANK3 mutant, overexpression prevents Rap1Q63E-mediated β1-integrin activation in HEK293 cells. Data represent mean ± SEM relative to mRFP and GFP expressing cells (n = 4 independent experiments; 5000 mRFP/GFP-positive cells analysed per experiment). b , c , Representative confocal images (b) and quantification (c) of spreading MDA-MB-231 cells transfected with GFP-Rap1Q63E alone or together with SHANK3 WT-mRFP. Staining: F-actin and DAPI (cell nuclei). Middle plane confocal image is shown. Scale bar: 10 μm. Tukey box plots represent median cell area relative to untransfected cells (n = 26, 21, 27 cells from left to right from three independent experiments). d,e , Representative confocal images (d) and quantification (e) of filopodia (indicated by arrowheads) in rat hippocampal neurons plated on laminin. Staining: F-actin and Map2 (neurite marker). Tukey box plots are shown (n = 176 (WT), 120 (L68P) from two independent experiments). Scale bar = 20 μm (original image) and 10 µm (ROI). f,g , Representative confocal images (f) and quantification (g) of filopodia in SK-N-BE-2 neuroblastoma cells plated on laminin and differentiated with retinoid acid (10 µM, three days). Shank3 SPN WT-GFP overexpression reduces filopodia density in neurites. Tukey box plots are shown (n = 76 (GFP), 62 (WT), three independent experiments). Scale bar = 20 μm. h , FACS analysis of β1-integrin activity in SHANK3- silenced HEK293 cells treated with a Rap1 inhibitor (10 μM, 1 h). (f) Data are mean ± SEM (n = 5 independent experiments; 10000 cells per experiment). i,j , Representative images (i) and Image J quantification (j) of cell area in SHANK3- silenced HEK293 cells ± Rap1 inhibitor (10 µM, 1 h). Cells were adhering on a fibronectin-collagen matrix (15 min). Middle plane confocal image is shown. Scale bar = 10 μm. Data are represented by Tukey box plots (n = 30, 33, 38 cells from left to right from two independent experiments. Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3 .

    Techniques Used: Activation Assay, FACS, Mutagenesis, Over Expression, Expressing, Multiple Displacement Amplification, Transfection, Staining, Marker, Activity Assay

    SHANK1 and SHANK3 regulate cell migration and invasion a-e , MDA-MB-231 cell migration on a fibronectin-collagen matrix recorded over 24 h by time-lapse imaging showing that SHANK1 -silenced cells migrate faster and more randomly than control-silenced cells (a-c). Re-expression of Shank1-GFP in SHANK1 -silenced cells rescued defects observed in cells migration (d, e). Representative cell tracks over 10 h (a) and quantification of the migration speed (b, d) and directionality (c, e) over 24 h are shown. Data were analysed using the Manual Tracking plugin (ImageJ) and are displayed as Tukey box plots (n = 36 (siCtrl) and 37 (siSHANK1) from three independent experiments (b and c); n = 36, 29, 28 from left to right from three independent experiments (d and e)). f , g , Inverted invasion assay showing increased MDA-MB-231 cell invasion upon SHANK1 silencing. Invasion in collagen plugs supplemented with fibronectin was visualized using a confocal microscope by imaging serial optical sections at 15 µm intervals. Individual confocal images are shown in sequence with increasing penetrance from left to right (f). Invasion was quantified using ImageJ by measuring the fluorescence intensity of cells invading 45 µm or more and expressing this as a ratio of the fluorescence intensity of all cells within the plug. Data are presented as Tukey box plots (g) (n = 6 means from three independent experiments). h , Schematic representation of SHANK-Rap1–dependent integrin inactivation regulating cell adhesion, spreading, migration and invasion. Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test.
    Figure Legend Snippet: SHANK1 and SHANK3 regulate cell migration and invasion a-e , MDA-MB-231 cell migration on a fibronectin-collagen matrix recorded over 24 h by time-lapse imaging showing that SHANK1 -silenced cells migrate faster and more randomly than control-silenced cells (a-c). Re-expression of Shank1-GFP in SHANK1 -silenced cells rescued defects observed in cells migration (d, e). Representative cell tracks over 10 h (a) and quantification of the migration speed (b, d) and directionality (c, e) over 24 h are shown. Data were analysed using the Manual Tracking plugin (ImageJ) and are displayed as Tukey box plots (n = 36 (siCtrl) and 37 (siSHANK1) from three independent experiments (b and c); n = 36, 29, 28 from left to right from three independent experiments (d and e)). f , g , Inverted invasion assay showing increased MDA-MB-231 cell invasion upon SHANK1 silencing. Invasion in collagen plugs supplemented with fibronectin was visualized using a confocal microscope by imaging serial optical sections at 15 µm intervals. Individual confocal images are shown in sequence with increasing penetrance from left to right (f). Invasion was quantified using ImageJ by measuring the fluorescence intensity of cells invading 45 µm or more and expressing this as a ratio of the fluorescence intensity of all cells within the plug. Data are presented as Tukey box plots (g) (n = 6 means from three independent experiments). h , Schematic representation of SHANK-Rap1–dependent integrin inactivation regulating cell adhesion, spreading, migration and invasion. Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test.

    Techniques Used: Migration, Multiple Displacement Amplification, Imaging, Expressing, Invasion Assay, Microscopy, Sequencing, Fluorescence

    SHANK1 and SHANK3 sequester active Rap1 to inhibit RIAM/talin-dependent integrin activation and alter adhesion dynamics a,b , Representative PLA signal (red dots) (a) and quantification of the distribution of the PLA signal relative to the total cell area (F-actin) (b) revealing more co-localization between talin and α5-integrin following SHANK3 silencing in HEK293 cells. Data are displayed as Tukey box plots and expressed relative to control-silenced cells (n = 72 cells from three independent experiments). c , β1-integrin (P5D2) immunoprecipitation showing enhanced interaction between β1-integrin and talin following SHANK3 silencing in HEK293 cells (representative blot from four independent experiments is shown). d,e , MDA-MB-231 cells transiently expressing GFP-Talin1 were plated on fibronectin-collagen and imaged live using a TIRF microscope (1 picture every 1 min for more than 3 h; scale bar = 20 μm (original image) and 10 µm (ROI)) (d). The percentage of cells displaying a “talin wave” was then counted (three biological repeats; 84 siCTRL and 86 siSHANK1 movies analysed) (e). f,g , MDA-MB-231 cells transiently expressing mEmerald-Paxillin were plated on fibronectin-collagen and imaged live using a TIRF microscope (1 picture every 1 min for more than 3 h; scale bar = 20 μm) (f). Focal adhesion lifetime was analysed using the focal adhesion analysis server (see methods ) (two biological repeats; over 33 movies per condition analysed; n = 97817 siCTRL and 164092 siSHANK1 adhesions analysed) (g). Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig 8 .
    Figure Legend Snippet: SHANK1 and SHANK3 sequester active Rap1 to inhibit RIAM/talin-dependent integrin activation and alter adhesion dynamics a,b , Representative PLA signal (red dots) (a) and quantification of the distribution of the PLA signal relative to the total cell area (F-actin) (b) revealing more co-localization between talin and α5-integrin following SHANK3 silencing in HEK293 cells. Data are displayed as Tukey box plots and expressed relative to control-silenced cells (n = 72 cells from three independent experiments). c , β1-integrin (P5D2) immunoprecipitation showing enhanced interaction between β1-integrin and talin following SHANK3 silencing in HEK293 cells (representative blot from four independent experiments is shown). d,e , MDA-MB-231 cells transiently expressing GFP-Talin1 were plated on fibronectin-collagen and imaged live using a TIRF microscope (1 picture every 1 min for more than 3 h; scale bar = 20 μm (original image) and 10 µm (ROI)) (d). The percentage of cells displaying a “talin wave” was then counted (three biological repeats; 84 siCTRL and 86 siSHANK1 movies analysed) (e). f,g , MDA-MB-231 cells transiently expressing mEmerald-Paxillin were plated on fibronectin-collagen and imaged live using a TIRF microscope (1 picture every 1 min for more than 3 h; scale bar = 20 μm) (f). Focal adhesion lifetime was analysed using the focal adhesion analysis server (see methods ) (two biological repeats; over 33 movies per condition analysed; n = 97817 siCTRL and 164092 siSHANK1 adhesions analysed) (g). Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig 8 .

    Techniques Used: Activation Assay, Proximity Ligation Assay, Immunoprecipitation, Multiple Displacement Amplification, Expressing, Microscopy

    Loss of SHANK1 and SHANK3 promotes cell adhesion and spreading a , Rate of cell adherence (cell index) of HEK293 cells monitored in real-time using the xCELLigence system shows enhanced cell attachment upon SHANK3 silencing on a fibronectin-collagen substrate. BSA was used as a control for background binding. Data represent mean ± SEM (n = 4 independent experiments; average of 4 wells per experiment, 20000 cells/well). b , Representative confocal images of control- and SHANK3- silenced HEK293 cells adhering to a fibronectin-collagen substrate for 15 min. Cells were stained for active β1-integrin (9EG7, green), paxillin (grey) and F-actin (phalloidin, red) to mark adhesions. Shown are confocal slices from the bottom surface. Scale bar = 10 μm (original image) and 5 µm (ROI). c-e , Quantification of the active integrin (9EG7-positive) adhesions in b showing that SHANK3 -silenced HEK293 cells form more (c) but smaller (d) adhesions as compared to control-silenced cells. The distribution of adhesion size is also shown (e). Adhesions were analysed using the Cell Profiler software. Data shown as Tukey box plots (n = 15 siCTRL cells and 22 siSHANK3 cells from 3 independent experiments). f,g , Representative confocal images (f) and quantification (g) showing increased HEK293 cell spreading on a fibronectin-collagen substrate upon SHANK3 silencing. Cells were stained for F-actin (phalloidin). Scale bar = 7 μm. Quantification of cell area at 20 or 60 min post-plating was analysed using Image J. Data displayed as Tukey box plots (n = 47 cells (20 min) or 72 cells (60 min) from 3 independent experiments). h , i , Representative confocal images (h) and quantification (i) showing increased HEK293 cell length upon SHANK3 silencing compared to control cells on fibronectin-collagen-coated micropattern lines at 20 or 60 min post-plating. Scale bar = 10 μm. Data are displayed as Tukey box plots (n = 29 cells (20 min) or 46 cells (60 min) from 3 independent experiments). Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3 .
    Figure Legend Snippet: Loss of SHANK1 and SHANK3 promotes cell adhesion and spreading a , Rate of cell adherence (cell index) of HEK293 cells monitored in real-time using the xCELLigence system shows enhanced cell attachment upon SHANK3 silencing on a fibronectin-collagen substrate. BSA was used as a control for background binding. Data represent mean ± SEM (n = 4 independent experiments; average of 4 wells per experiment, 20000 cells/well). b , Representative confocal images of control- and SHANK3- silenced HEK293 cells adhering to a fibronectin-collagen substrate for 15 min. Cells were stained for active β1-integrin (9EG7, green), paxillin (grey) and F-actin (phalloidin, red) to mark adhesions. Shown are confocal slices from the bottom surface. Scale bar = 10 μm (original image) and 5 µm (ROI). c-e , Quantification of the active integrin (9EG7-positive) adhesions in b showing that SHANK3 -silenced HEK293 cells form more (c) but smaller (d) adhesions as compared to control-silenced cells. The distribution of adhesion size is also shown (e). Adhesions were analysed using the Cell Profiler software. Data shown as Tukey box plots (n = 15 siCTRL cells and 22 siSHANK3 cells from 3 independent experiments). f,g , Representative confocal images (f) and quantification (g) showing increased HEK293 cell spreading on a fibronectin-collagen substrate upon SHANK3 silencing. Cells were stained for F-actin (phalloidin). Scale bar = 7 μm. Quantification of cell area at 20 or 60 min post-plating was analysed using Image J. Data displayed as Tukey box plots (n = 47 cells (20 min) or 72 cells (60 min) from 3 independent experiments). h , i , Representative confocal images (h) and quantification (i) showing increased HEK293 cell length upon SHANK3 silencing compared to control cells on fibronectin-collagen-coated micropattern lines at 20 or 60 min post-plating. Scale bar = 10 μm. Data are displayed as Tukey box plots (n = 29 cells (20 min) or 46 cells (60 min) from 3 independent experiments). Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3 .

    Techniques Used: Cell Attachment Assay, Binding Assay, Staining, Software

    SHANK1 and SHANK3 inhibit β1-integrin activation a , Hierarchical clustering of β1-integrin activity (9EG7 and/or 12G10 antibodies; red: increased and blue: decreased compared to control-silenced cells (Z-score)) in 13 human cell lines upon SHANK1 or SHANK3 silencing with two independent siRNAs (#1 or #2). Results taken from a high-density cell-spot microarray. b , SHANK3 gene expression (log 10 RPKM: Reads Per Kilobase of transcript per Million mapped reads) in human tissues analysed using the publicly available GTEx portal (Grey region: brain tissues). c-e , Flow cytometric (FACS) analysis of integrin activity in the indicated conditions. c , Quantification shows reduced active cell-surface integrin (FN 7-10 binding) relative to total cell-surface α5β1-integrin (PB1 antibody) in Shank3-mRFP- or SHARPIN-GFP-expressing cells compared to mRFP/GFP cells. d , SHANK3 -silencing triggers β1-integrin activation (active β1-integrin: 9EG7 antibody; total β1-integrin: P5D2) similarly to Mn 2+ . e , Shank3-mRFP re-expression abrogates integrin activation induced by SHANK3 silencing. Data represent mean ± SEM (n = 5 (c), 3 (d), 4 (e) independent experiments; 5000 (mRFP- or GFP-positive cells) or 10000 cells ( SHANK 3-silenced) per experiment). f , FACS analysis (active β1-integrin: 9EG7 antibody; total β1-integrin: MAB1997) in MMECs isolated from Shank3αβ −/− mice compared to Shank3αβ +/+ (mean of 2 independent experiments; cells pooled from three mice per experiment). g , Shank3-mRFP-expressing MDA-MB-231 cells plated on fibronectin-collagen demonstrate SHANK3 localization with inactive β1-integrin (MAB13) and membrane marker CAAX-GFP in membrane ruffles. Shown is a representative confocal slice (middle plane). ROI: region of interest. Scale bar = 20 μm (original image) and 10 µm (ROI). h , HEK293 subcellular fractions. Cyt: cytoplasmic; PM: plasma membrane; Na + /K + pump: PM marker; tubulin: Cyt marker; 10 % Lys: 10 % of total lysate. i , Shank3-mRFP-expressing MDA-MB-231 cells plated on fibronectin and imaged live using a spinning disk microscope (1 picture every 10 s). Scale bar = 20 μm (original image) and 5 µm (ROI). Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3 . Unprocessed original scans of blots are shown in Supplementary Fig 8 .
    Figure Legend Snippet: SHANK1 and SHANK3 inhibit β1-integrin activation a , Hierarchical clustering of β1-integrin activity (9EG7 and/or 12G10 antibodies; red: increased and blue: decreased compared to control-silenced cells (Z-score)) in 13 human cell lines upon SHANK1 or SHANK3 silencing with two independent siRNAs (#1 or #2). Results taken from a high-density cell-spot microarray. b , SHANK3 gene expression (log 10 RPKM: Reads Per Kilobase of transcript per Million mapped reads) in human tissues analysed using the publicly available GTEx portal (Grey region: brain tissues). c-e , Flow cytometric (FACS) analysis of integrin activity in the indicated conditions. c , Quantification shows reduced active cell-surface integrin (FN 7-10 binding) relative to total cell-surface α5β1-integrin (PB1 antibody) in Shank3-mRFP- or SHARPIN-GFP-expressing cells compared to mRFP/GFP cells. d , SHANK3 -silencing triggers β1-integrin activation (active β1-integrin: 9EG7 antibody; total β1-integrin: P5D2) similarly to Mn 2+ . e , Shank3-mRFP re-expression abrogates integrin activation induced by SHANK3 silencing. Data represent mean ± SEM (n = 5 (c), 3 (d), 4 (e) independent experiments; 5000 (mRFP- or GFP-positive cells) or 10000 cells ( SHANK 3-silenced) per experiment). f , FACS analysis (active β1-integrin: 9EG7 antibody; total β1-integrin: MAB1997) in MMECs isolated from Shank3αβ −/− mice compared to Shank3αβ +/+ (mean of 2 independent experiments; cells pooled from three mice per experiment). g , Shank3-mRFP-expressing MDA-MB-231 cells plated on fibronectin-collagen demonstrate SHANK3 localization with inactive β1-integrin (MAB13) and membrane marker CAAX-GFP in membrane ruffles. Shown is a representative confocal slice (middle plane). ROI: region of interest. Scale bar = 20 μm (original image) and 10 µm (ROI). h , HEK293 subcellular fractions. Cyt: cytoplasmic; PM: plasma membrane; Na + /K + pump: PM marker; tubulin: Cyt marker; 10 % Lys: 10 % of total lysate. i , Shank3-mRFP-expressing MDA-MB-231 cells plated on fibronectin and imaged live using a spinning disk microscope (1 picture every 10 s). Scale bar = 20 μm (original image) and 5 µm (ROI). Tukey box plots represent median and 25 th and 75 th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3 . Unprocessed original scans of blots are shown in Supplementary Fig 8 .

    Techniques Used: Activation Assay, Activity Assay, Microarray, Expressing, Flow Cytometry, FACS, Binding Assay, Isolation, Mouse Assay, Multiple Displacement Amplification, Marker, Microscopy

    11) Product Images from "Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model"

    Article Title: Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0187109

    Anti-cadherin-11 antibody decreases mediators of fibrosis in Tsk-1 mice. Total RNA was isolated from skin biopsies from pa/pa control mice, Tsk-1 mice treated with isotype antibodies, and Tsk-1 mice treated with antibodies against CDH11 (13C2). Transcripts were determined for Col1 α 1 (a), α SMA (b), CCN2 (c), fibronectin (d), TGF- β (e), IL-6 (f) in parallel with 18S rRNA. Data are presented as mean of fold change transcripts ± SEM, n≥10 (*p ≤ 0.05 pa/pa vs. Tsk-1 isotype; #p ≤ 0.05 Tsk-1 isotype vs. Tsk-1 anti-CDH11).
    Figure Legend Snippet: Anti-cadherin-11 antibody decreases mediators of fibrosis in Tsk-1 mice. Total RNA was isolated from skin biopsies from pa/pa control mice, Tsk-1 mice treated with isotype antibodies, and Tsk-1 mice treated with antibodies against CDH11 (13C2). Transcripts were determined for Col1 α 1 (a), α SMA (b), CCN2 (c), fibronectin (d), TGF- β (e), IL-6 (f) in parallel with 18S rRNA. Data are presented as mean of fold change transcripts ± SEM, n≥10 (*p ≤ 0.05 pa/pa vs. Tsk-1 isotype; #p ≤ 0.05 Tsk-1 isotype vs. Tsk-1 anti-CDH11).

    Techniques Used: Mouse Assay, Isolation

    12) Product Images from "Telmisartan in the diabetic murine model of acute myocardial infarction: dual contrast manganese-enhanced and delayed enhancement MRI evaluation of the peri-infarct region"

    Article Title: Telmisartan in the diabetic murine model of acute myocardial infarction: dual contrast manganese-enhanced and delayed enhancement MRI evaluation of the peri-infarct region

    Journal: Cardiovascular Diabetology

    doi: 10.1186/s12933-016-0348-y

    Myocardial fibrosis and apoptosis. Real-time PCR quantitative analysis of fibrotic (collagen I, collagen III, connective tissue growth factor (CTGF), TGFβ, and fibronectin) and apoptotic (thymoma viral proto-oncogene 1: Akt) genes in the injured myocardium of telmisartan and control groups. There was a trend towards increased expression of fibrotic genes, collagen I, collagen III, CTGF and fibronectin in the telmisartan group compared to the control group. Red bar : telmisartan group, white bar : control group
    Figure Legend Snippet: Myocardial fibrosis and apoptosis. Real-time PCR quantitative analysis of fibrotic (collagen I, collagen III, connective tissue growth factor (CTGF), TGFβ, and fibronectin) and apoptotic (thymoma viral proto-oncogene 1: Akt) genes in the injured myocardium of telmisartan and control groups. There was a trend towards increased expression of fibrotic genes, collagen I, collagen III, CTGF and fibronectin in the telmisartan group compared to the control group. Red bar : telmisartan group, white bar : control group

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    13) Product Images from "NMDA-Dependent Proteolysis of Presynaptic Adhesion Molecule L1 in the Hippocampus by Neuropsin"

    Article Title: NMDA-Dependent Proteolysis of Presynaptic Adhesion Molecule L1 in the Hippocampus by Neuropsin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.23-21-07727.2003

    The proteolytic effects of neuropsin on CAMs and ECM proteins. A , An electron micrograph representing the prepared SN fraction. The synaptoneurosome consists of functional resealed presynaptic (Pre) plus postsynaptic (Post) entities. Scale bar, 0.5 μm. * indicates the synaptic adhesive site. B-E , Immunoblot analysis of the SN fractions treated for 60 min with a solvent (Buf), r-proNP (Pro), and r-actNP (Act) using anti-NCAM antibody ( B ), anti-L1 antibody ( C ), anti-E-cadherin antibody ( D ), and anti-N-cadherin antibody ( E ). Application of r-actNP resulted in a complete loss of intact 200 kDa L1 and a marked reduction in its 80 kDa C-terminal fragment ( C ). In contrast, NCAM ( B ), E-cadherin ( D ), and N-cadherin ( E ) were not affected by coincubation with r-actNP. F, G , Coomassie blue staining of laminin ( F ) and fibronectin ( G ) applied for 60 min with buffer only (Buf), r-proNP (Pro), and r-actNP (Act). The application of r-actNP with fibronectin resulted in a decrease in the intact 220 kDa band and the appearance of 215, 210, and 200 kDa fragments ( G ). In contrast, r-actNP was less effective against laminin ( F ). H , The time course in cleavage of CAMs and ECM molecules induced by coincubation with r-actNP. The band densities were quantified by densito-metric scanning setting densities in the SN fraction before application of r-actNP (0 min) as 100%, and the relative densities (vertical axis) were plotted at various time points after the application of r-actNP (horizontal axis). All data are presented as the mean ± SEM of two independent experiments. Of these molecules, L1 showed the highest velocity of cleavage. The 200 kDa band density of intact L1 was reduced to one-half at 15 min.
    Figure Legend Snippet: The proteolytic effects of neuropsin on CAMs and ECM proteins. A , An electron micrograph representing the prepared SN fraction. The synaptoneurosome consists of functional resealed presynaptic (Pre) plus postsynaptic (Post) entities. Scale bar, 0.5 μm. * indicates the synaptic adhesive site. B-E , Immunoblot analysis of the SN fractions treated for 60 min with a solvent (Buf), r-proNP (Pro), and r-actNP (Act) using anti-NCAM antibody ( B ), anti-L1 antibody ( C ), anti-E-cadherin antibody ( D ), and anti-N-cadherin antibody ( E ). Application of r-actNP resulted in a complete loss of intact 200 kDa L1 and a marked reduction in its 80 kDa C-terminal fragment ( C ). In contrast, NCAM ( B ), E-cadherin ( D ), and N-cadherin ( E ) were not affected by coincubation with r-actNP. F, G , Coomassie blue staining of laminin ( F ) and fibronectin ( G ) applied for 60 min with buffer only (Buf), r-proNP (Pro), and r-actNP (Act). The application of r-actNP with fibronectin resulted in a decrease in the intact 220 kDa band and the appearance of 215, 210, and 200 kDa fragments ( G ). In contrast, r-actNP was less effective against laminin ( F ). H , The time course in cleavage of CAMs and ECM molecules induced by coincubation with r-actNP. The band densities were quantified by densito-metric scanning setting densities in the SN fraction before application of r-actNP (0 min) as 100%, and the relative densities (vertical axis) were plotted at various time points after the application of r-actNP (horizontal axis). All data are presented as the mean ± SEM of two independent experiments. Of these molecules, L1 showed the highest velocity of cleavage. The 200 kDa band density of intact L1 was reduced to one-half at 15 min.

    Techniques Used: Functional Assay, Activated Clotting Time Assay, Staining

    Chemical stimulation induced neuropsin-dependent L1 cleavage. A , Immunoblot analysis showed that L1-180 (arrowheads) increased in the hippocampal organs stimulated by 4-AP or NMDA but not in the absence of stimulation (NS) or in the neuropsin-deficient hippocampus stimulated with NMDA [NMDA (NP-/-)] at both 15 and 30 min after treatment. The 4-AP-induced or NMDA-induced increase of L1-180 was blocked by preincubation with AP-5 (4-AP + AP5) or anti-neuropsin antibody mAbB5 (NMDA + mAbB5). B , Temporal change in the L1-180 band density (top) and the band density ratio of L1-180/L1-200 (bottom graph) after chemical stimulation. Error bars indicate SEM. A rapid neuropsin-dependent increase in L1-180 was induced by chemical stimulation with 4-AP and NMDA. C, D , Immunoblot analyses using anti-N-cadherin ( C ) and anti-fibronectin ( D ) antibodies revealed that no change in N-cadherin and fibronectin in the hippocampal organs was induced by chemical stimulation (4-AP or NMDA).
    Figure Legend Snippet: Chemical stimulation induced neuropsin-dependent L1 cleavage. A , Immunoblot analysis showed that L1-180 (arrowheads) increased in the hippocampal organs stimulated by 4-AP or NMDA but not in the absence of stimulation (NS) or in the neuropsin-deficient hippocampus stimulated with NMDA [NMDA (NP-/-)] at both 15 and 30 min after treatment. The 4-AP-induced or NMDA-induced increase of L1-180 was blocked by preincubation with AP-5 (4-AP + AP5) or anti-neuropsin antibody mAbB5 (NMDA + mAbB5). B , Temporal change in the L1-180 band density (top) and the band density ratio of L1-180/L1-200 (bottom graph) after chemical stimulation. Error bars indicate SEM. A rapid neuropsin-dependent increase in L1-180 was induced by chemical stimulation with 4-AP and NMDA. C, D , Immunoblot analyses using anti-N-cadherin ( C ) and anti-fibronectin ( D ) antibodies revealed that no change in N-cadherin and fibronectin in the hippocampal organs was induced by chemical stimulation (4-AP or NMDA).

    Techniques Used:

    14) Product Images from "Cadherin-11 localizes to focal adhesions and promotes cell–substrate adhesion"

    Article Title: Cadherin-11 localizes to focal adhesions and promotes cell–substrate adhesion

    Journal: Nature Communications

    doi: 10.1038/ncomms10909

    Xcad-11 is localized in focal adhesions. Xenopus NCC injected with Xcad-11-EGFP, explanted on fibronectin-coated glass dishes and immunostained for ( a ) β-catenin, ( b ) paxillin and ( c ) β1-integrin. ( a ) A confocal image focused on the apical side of NCC shows co-localization of Xcad-11 with β-catenin at cell–cell contacts. ( b , c ) TIRF images demonstrating co-localization of Xcad-11 with paxillin and β1-integrin in focal adhesions at the cell substrate. ( d ) HeLa cells transfected with Xcad-11-EGFP, immunostained for paxillin and imaged by TIRF microscopy display partial localization of Xcad-11 with paxillin at the cell substrate. Scale bars, 20 μm ( a ); 10 μm ( b – d ).
    Figure Legend Snippet: Xcad-11 is localized in focal adhesions. Xenopus NCC injected with Xcad-11-EGFP, explanted on fibronectin-coated glass dishes and immunostained for ( a ) β-catenin, ( b ) paxillin and ( c ) β1-integrin. ( a ) A confocal image focused on the apical side of NCC shows co-localization of Xcad-11 with β-catenin at cell–cell contacts. ( b , c ) TIRF images demonstrating co-localization of Xcad-11 with paxillin and β1-integrin in focal adhesions at the cell substrate. ( d ) HeLa cells transfected with Xcad-11-EGFP, immunostained for paxillin and imaged by TIRF microscopy display partial localization of Xcad-11 with paxillin at the cell substrate. Scale bars, 20 μm ( a ); 10 μm ( b – d ).

    Techniques Used: Injection, Transfection, Microscopy

    15) Product Images from "Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes"

    Article Title: Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes

    Journal: Molecular Biology of the Cell

    doi:

    Analysis of recombinant chick tenascin-C proteins. (A) SDS-PAGE analysis. Lane 1, molecular mass standards (from the top), 200 kDa, 116 kDa, 97 kDa, and 66 kDa; lane 2, recombinant tenascin-C variant that includes the fibronectin type III repeats AD2, AD1, and C (TN-ADC); lane 3, recombinant tenascin-190 (TN-190); lane 4, tenascin-C from chicken embryo fibroblast (CEF-TN; this contains a mixture of TN-190, TN-200, and TN-230 protein variants). All samples were resolved on a 6% polyacrylamide gel under reducing conditions. (B and C) Electron microscopy of recombinant tenascin-Cs. TN -190 (B) or TN-ADC (C) were sprayed onto mica and rotary-shadowed. Bar, 50 nm. (D) Schematic diagram showing ordering of the variable fibronectin type III repeats in chicken (top) and the repeat combinations in CEF-TN, TN-190, or TN-ADC proteins.
    Figure Legend Snippet: Analysis of recombinant chick tenascin-C proteins. (A) SDS-PAGE analysis. Lane 1, molecular mass standards (from the top), 200 kDa, 116 kDa, 97 kDa, and 66 kDa; lane 2, recombinant tenascin-C variant that includes the fibronectin type III repeats AD2, AD1, and C (TN-ADC); lane 3, recombinant tenascin-190 (TN-190); lane 4, tenascin-C from chicken embryo fibroblast (CEF-TN; this contains a mixture of TN-190, TN-200, and TN-230 protein variants). All samples were resolved on a 6% polyacrylamide gel under reducing conditions. (B and C) Electron microscopy of recombinant tenascin-Cs. TN -190 (B) or TN-ADC (C) were sprayed onto mica and rotary-shadowed. Bar, 50 nm. (D) Schematic diagram showing ordering of the variable fibronectin type III repeats in chicken (top) and the repeat combinations in CEF-TN, TN-190, or TN-ADC proteins.

    Techniques Used: Recombinant, SDS Page, Variant Assay, Electron Microscopy

    Presence of fascin-positive microspikes in cells adherent on tenascin-C substrata. C2C12 (a, c, e, and g) or S27 (b, d, f, and h) cells were stained for fascin after a 90-min adhesion to fibronectin (a and b), CEF-TN (c and d), TN-190 (e and f), or TN-ADC (g and h). The arrays of microspikes formed by cells adherent on the tenascin-C substrata stain positively for fascin (examples are indicated by arrows in c, d, f, and h). Bar, 10 μm.
    Figure Legend Snippet: Presence of fascin-positive microspikes in cells adherent on tenascin-C substrata. C2C12 (a, c, e, and g) or S27 (b, d, f, and h) cells were stained for fascin after a 90-min adhesion to fibronectin (a and b), CEF-TN (c and d), TN-190 (e and f), or TN-ADC (g and h). The arrays of microspikes formed by cells adherent on the tenascin-C substrata stain positively for fascin (examples are indicated by arrows in c, d, f, and h). Bar, 10 μm.

    Techniques Used: Staining

    Localization of tenascin-C and fibronectin in sternum and keel. Cross-sections through sternum and keel of an E10 chick were processed for immunohistochemistry (A and B) or in situ hybridization (C–G). (A) Antibody to tenascin-C stains the perichondrium surrounding the sternal anlages (s) and the keel (k) intensely. (B) Antibody to cellular fibronectin stains the fusion point between the two sternal anlage (arrowhead), matrix surrounding the pectoral muscles (p) and the keel (k). (C) Universal probe TN-EGF hybridizes in sternal perichondrium (arrowhead) and in the keel (k). (D) AD1 probe. (E) AD2 probe. (F) C probe. All three probes hybridize in the keel. (G) pUC negative control. Bar, 100 μm.
    Figure Legend Snippet: Localization of tenascin-C and fibronectin in sternum and keel. Cross-sections through sternum and keel of an E10 chick were processed for immunohistochemistry (A and B) or in situ hybridization (C–G). (A) Antibody to tenascin-C stains the perichondrium surrounding the sternal anlages (s) and the keel (k) intensely. (B) Antibody to cellular fibronectin stains the fusion point between the two sternal anlage (arrowhead), matrix surrounding the pectoral muscles (p) and the keel (k). (C) Universal probe TN-EGF hybridizes in sternal perichondrium (arrowhead) and in the keel (k). (D) AD1 probe. (E) AD2 probe. (F) C probe. All three probes hybridize in the keel. (G) pUC negative control. Bar, 100 μm.

    Techniques Used: Immunohistochemistry, In Situ Hybridization, Negative Control

    Cytoskeletal reorganization in response to soluble tenascin-Cs. C2C12 cells (a–h), COS-7 cells (i–l), or MG-63 cells (m–p) adherent on fibronectin (a, e, i, and m), on fibronectin in the presence of 35 nM CEF-TN (b, f, j, and n), in the presence of 35 nM TN-190 (c, g, k, and o), or in the presence of 35 nM TN-ADC (d, h, l, and p) were stained after 1 h with TRITC-phalloidin (e–p) or for fascin (a–d). Bar, 15 μm.
    Figure Legend Snippet: Cytoskeletal reorganization in response to soluble tenascin-Cs. C2C12 cells (a–h), COS-7 cells (i–l), or MG-63 cells (m–p) adherent on fibronectin (a, e, i, and m), on fibronectin in the presence of 35 nM CEF-TN (b, f, j, and n), in the presence of 35 nM TN-190 (c, g, k, and o), or in the presence of 35 nM TN-ADC (d, h, l, and p) were stained after 1 h with TRITC-phalloidin (e–p) or for fascin (a–d). Bar, 15 μm.

    Techniques Used: Staining

    Cells adherent on tenascin-C substrata do not assemble focal contacts. C2C12 (a and c) or S27 (b and d) cells were stained for vinculin after a 90-min adhesion to fibronectin (a and b) or CEF-TN (c and d). Focal contacts are present in the cells on fibronectin, but the cytoplasmic extensions and microspikes of cells adherent on CEF-TN show no localization of vinculin (small arrows in c and d indicate positions of protrusions). Bar, 10 μm.
    Figure Legend Snippet: Cells adherent on tenascin-C substrata do not assemble focal contacts. C2C12 (a and c) or S27 (b and d) cells were stained for vinculin after a 90-min adhesion to fibronectin (a and b) or CEF-TN (c and d). Focal contacts are present in the cells on fibronectin, but the cytoplasmic extensions and microspikes of cells adherent on CEF-TN show no localization of vinculin (small arrows in c and d indicate positions of protrusions). Bar, 10 μm.

    Techniques Used: Staining

    F-actin and fascin distributions in C2C12 cells adherent on low concentrations of fibronectin. C2C12 cells were allowed to attach for 1 h to substrata coated with 1.25 μg/ml (A and B), 2.5 μg/ml (C and D), 5 μg/ml (E and F), or 20 μg/ml fibronectin (G and H) for 1 h then fixed and stained with TRITC-phalloidin (A, C, E, and G) or with antibody to fascin (B, D, F, and H). Arrows in A–D indicate examples of cells with F-actin- or fascin-containing membrane protrusions. Bar, 10 μm.
    Figure Legend Snippet: F-actin and fascin distributions in C2C12 cells adherent on low concentrations of fibronectin. C2C12 cells were allowed to attach for 1 h to substrata coated with 1.25 μg/ml (A and B), 2.5 μg/ml (C and D), 5 μg/ml (E and F), or 20 μg/ml fibronectin (G and H) for 1 h then fixed and stained with TRITC-phalloidin (A, C, E, and G) or with antibody to fascin (B, D, F, and H). Arrows in A–D indicate examples of cells with F-actin- or fascin-containing membrane protrusions. Bar, 10 μm.

    Techniques Used: Staining

    Organization of actin microfilaments in S27 and G8 cells adherent on fibronectin or tenascin-C. Cells were plated onto coverslips coated with 50 nM fibronectin, 50 nM CEF-TN, 50 nM recombinant TN-190, or 50 nM recombinant TN-ADC for 90 min in serum-free medium. After incubation cells were fixed, stained with TRITC-phalloidin, and examined under epifluorescence. Bar, 10 μm.
    Figure Legend Snippet: Organization of actin microfilaments in S27 and G8 cells adherent on fibronectin or tenascin-C. Cells were plated onto coverslips coated with 50 nM fibronectin, 50 nM CEF-TN, 50 nM recombinant TN-190, or 50 nM recombinant TN-ADC for 90 min in serum-free medium. After incubation cells were fixed, stained with TRITC-phalloidin, and examined under epifluorescence. Bar, 10 μm.

    Techniques Used: Recombinant, Incubation, Staining

    Confocal microscopy of actin microfilament organization in C2C12 and S27 cells adherent on fibronectin or tenascin-C. Cells were plated onto coverslips coated with 50 nM fibronectin (a and b), 50 nM CEF-TN (c and d), 50 nM recombinant TN-190 (e and f), or 50 nM recombinant TN-ADC (g and h) for 90 min in serum-free medium. After incubation cells were fixed, stained with TRITC-phalloidin, and photographed on a confocal microscope. Bar, 5 μm.
    Figure Legend Snippet: Confocal microscopy of actin microfilament organization in C2C12 and S27 cells adherent on fibronectin or tenascin-C. Cells were plated onto coverslips coated with 50 nM fibronectin (a and b), 50 nM CEF-TN (c and d), 50 nM recombinant TN-190 (e and f), or 50 nM recombinant TN-ADC (g and h) for 90 min in serum-free medium. After incubation cells were fixed, stained with TRITC-phalloidin, and photographed on a confocal microscope. Bar, 5 μm.

    Techniques Used: Confocal Microscopy, Recombinant, Incubation, Staining, Microscopy

    Localization of tenascin-C and fibronectin in feather buds. (A) Antibody to tenascin-C stains the mesenchyme at the base of the feather buds. (B) Antibody to cellular fibronectin stains all of the dermal extracellular matrix and the underlying blood vessels. (C–F) In situ hybridization on adjacent cross-sections through the dorsal feather tract of an E10 chick. (C) Tenascin C probe specific to fibronectin type III repeat AD1. (D) Universal tenascin-C probe TN-EGF. (E) Probe to repeat AD2. (F) pUC negative control probe. All the tenascin-C probes hybridize in the mesenchyme at the base of the feather buds. Bar, 100 μm. (G) RT-PCR detection of AD2AD1C-containing tenascin-C transcript in embryonic tissues. Primers corresponding to the beginning of repeat AD2 and the end of repeat C were used to amplify products from mRNA isolated from E10 chicken lung (lane 1) or wing (lane 2). Lane 3 was loaded with PCR product corresponding to repeat C. The blot was probed with cAD2. A single band of about 550 bp (i.e., two fibronectin type III repeats) was detected in amplification products from lung. In contrast, two bands corresponding to products containing 2 and 3 fibronectin type III repeats were detected in wingbud (arrows). DNA standards from bottom to top are 100 bp, 200 bp, 300 bp, 400 bp, 600 bp, 800 bp, and 2000 bp.
    Figure Legend Snippet: Localization of tenascin-C and fibronectin in feather buds. (A) Antibody to tenascin-C stains the mesenchyme at the base of the feather buds. (B) Antibody to cellular fibronectin stains all of the dermal extracellular matrix and the underlying blood vessels. (C–F) In situ hybridization on adjacent cross-sections through the dorsal feather tract of an E10 chick. (C) Tenascin C probe specific to fibronectin type III repeat AD1. (D) Universal tenascin-C probe TN-EGF. (E) Probe to repeat AD2. (F) pUC negative control probe. All the tenascin-C probes hybridize in the mesenchyme at the base of the feather buds. Bar, 100 μm. (G) RT-PCR detection of AD2AD1C-containing tenascin-C transcript in embryonic tissues. Primers corresponding to the beginning of repeat AD2 and the end of repeat C were used to amplify products from mRNA isolated from E10 chicken lung (lane 1) or wing (lane 2). Lane 3 was loaded with PCR product corresponding to repeat C. The blot was probed with cAD2. A single band of about 550 bp (i.e., two fibronectin type III repeats) was detected in amplification products from lung. In contrast, two bands corresponding to products containing 2 and 3 fibronectin type III repeats were detected in wingbud (arrows). DNA standards from bottom to top are 100 bp, 200 bp, 300 bp, 400 bp, 600 bp, 800 bp, and 2000 bp.

    Techniques Used: In Situ Hybridization, Negative Control, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification

    Modulation of cell attachment to fibronectin by tenascin-Cs in solution. C2C12, MG63, or COS-7 cells were allowed to attach to fibronectin substrata for 60 min in the absence or presence of 35 nM tenascin-C added in solution, as indicated, and the percentage of input cells that attached was quantitated by direct counting. Each column is the mean of three assays; bars are the SEM.
    Figure Legend Snippet: Modulation of cell attachment to fibronectin by tenascin-Cs in solution. C2C12, MG63, or COS-7 cells were allowed to attach to fibronectin substrata for 60 min in the absence or presence of 35 nM tenascin-C added in solution, as indicated, and the percentage of input cells that attached was quantitated by direct counting. Each column is the mean of three assays; bars are the SEM.

    Techniques Used: Cell Attachment Assay

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    Article Title: CTIP2 Associates with the NuRD Complex on the Promoter of p57KIP2, a Newly Identified CTIP2 Target Gene *
    Article Snippet: These genes are heme oxygenase-1 (HMOX-1), fibronectin-1 (FN-1), cadherin-10, and p57KIP2.

    Incubation:

    Article Title: Extracellular Acidic pH Inhibits Oligodendrocyte Precursor Viability, Migration, and Differentiation
    Article Snippet: The PDL-coated dishes were then incubated with laminin (mouse natural laminin-1, Invitrogen) for 1 h at 37°C, at different concentrations depending on the experiment (10–200 µg/mL), then washed twice with phosphate buffered saline at pH 7.4 (PBS; Gibco). .. Fibronectin Glass-bottom dishes were incubated with 10 µg/ml fibronectin (bovine plasma, Invitrogen) for 1 h at 37°C, then washed twice with PBS. .. Immunocytochemistry The primary antibodies used for immunocytochemistry were rat anti-MBP (Serotec) used to measure OPC differentiation, rabbit anti-Ki67 (Millipore) used to measure OPC proliferation, and mouse anti-integrin α6 β1 (Millipore) used to measure integrin expression in cells by flow cytometry.

    Purification:

    Article Title: Metalloproteinase-mediated Shedding of Integrin \u03b22 Promotes Macrophage Efflux from Inflammatory Sites
    Article Snippet: .. Polysorb microtiter plates were coated with different amounts of purified human fibrinogen (American Diagnostica), bovine collagen type I (Cohesion), and fibronectin (Invitrogen). .. For fibrinogen, bovine thrombin (Innovative Research; 6 μl of solution at 1.2 mg/ml, activity 1900 NIH units/mg) was added at 4 °C and left at RT for 30 min to allow fibrin formation.

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    Thermo Fisher mouse plasma fibronectin
    Adhesion and migration of PS explant cells in Boyden chambers. PS explants from α4-positive (A–D) or -null (E–F) embryos were placed on membranes coated with VCAM-1 (A and B) or <t>fibronectin</t> (C–F). After overnight incubation, each explant was photographed (A, C, and E) and removed from the top side; the bottom side was then photographed (B, D, and F). Note the nuclei of explant cells that adhered to VCAM-1 and fibronectin (A, C, and E) and migrated through pores (B and D, arrows). α4-null explant cells failed to migrate through pores (F, arrow). FN, fibronectin. Bar, 0.1 mm.
    Mouse Plasma Fibronectin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fibronectin coated dishes
    hGAAP expression alters the number and size of focal adhesions. U2-OS cells transfected with siRNA (24 h) or plasmids encoding hGAAP were seeded onto <t>fibronectin-coated</t> slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P
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    Thermo Fisher human fibronectin
    Flow chart for the protocol of simultaneous isolation of MSCs and EPCs derived from murine BM. BM cells were flushed out of femurs and tibias with DMEM complete medium. To obtain MSCs, the medium in culture dish A was gradually replaced with 1.5 ml fresh DMEM complete medium was performed every 8 h for 72 h. To obtain EPCs, non-adherent cells were plated in culture dish C, which was coated with human <t>fibronectin,</t> and maintained in endothelial growth medium. MSCs, mesenchymal stem or stromal cells; EPCs, endothelial progenitor cells; BM, bone marrow; DMEM, Dulbecco's modified Eagle's medium.
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    Image Search Results


    Adhesion and migration of PS explant cells in Boyden chambers. PS explants from α4-positive (A–D) or -null (E–F) embryos were placed on membranes coated with VCAM-1 (A and B) or fibronectin (C–F). After overnight incubation, each explant was photographed (A, C, and E) and removed from the top side; the bottom side was then photographed (B, D, and F). Note the nuclei of explant cells that adhered to VCAM-1 and fibronectin (A, C, and E) and migrated through pores (B and D, arrows). α4-null explant cells failed to migrate through pores (F, arrow). FN, fibronectin. Bar, 0.1 mm.

    Journal: The Journal of Cell Biology

    Article Title: Dual functions of ?4?1 integrin in epicardial development

    doi: 10.1083/jcb.200203075

    Figure Lengend Snippet: Adhesion and migration of PS explant cells in Boyden chambers. PS explants from α4-positive (A–D) or -null (E–F) embryos were placed on membranes coated with VCAM-1 (A and B) or fibronectin (C–F). After overnight incubation, each explant was photographed (A, C, and E) and removed from the top side; the bottom side was then photographed (B, D, and F). Note the nuclei of explant cells that adhered to VCAM-1 and fibronectin (A, C, and E) and migrated through pores (B and D, arrows). α4-null explant cells failed to migrate through pores (F, arrow). FN, fibronectin. Bar, 0.1 mm.

    Article Snippet: 10 μg/ml mouse plasma fibronectin (Life Technologies) or soluble VCAM-1, provided by Roy Lobb (Biogene, Cambridge, MA) in DME were added to the bottom compartments of the chambers.

    Techniques: Migration, Incubation

    Fibronectin and VCAM-1 localization by immunostaining. Embryo sections were stained with an anti-fibronectin (A and C) or an anti–VCAM-1 antibody (B and D). C and D are higher magnifications of A and B, respectively, showing the PS region. Note that fibronectin is localized in the periphery of cysts that are budding out (arrowhead) and in the myocardium. VCAM-1 is localized in the myocardium. a, atrium; v, ventricle; my, myocardium. Bars, 0.1 mm.

    Journal: The Journal of Cell Biology

    Article Title: Dual functions of ?4?1 integrin in epicardial development

    doi: 10.1083/jcb.200203075

    Figure Lengend Snippet: Fibronectin and VCAM-1 localization by immunostaining. Embryo sections were stained with an anti-fibronectin (A and C) or an anti–VCAM-1 antibody (B and D). C and D are higher magnifications of A and B, respectively, showing the PS region. Note that fibronectin is localized in the periphery of cysts that are budding out (arrowhead) and in the myocardium. VCAM-1 is localized in the myocardium. a, atrium; v, ventricle; my, myocardium. Bars, 0.1 mm.

    Article Snippet: 10 μg/ml mouse plasma fibronectin (Life Technologies) or soluble VCAM-1, provided by Roy Lobb (Biogene, Cambridge, MA) in DME were added to the bottom compartments of the chambers.

    Techniques: Immunostaining, Staining

    hGAAP expression alters the number and size of focal adhesions. U2-OS cells transfected with siRNA (24 h) or plasmids encoding hGAAP were seeded onto fibronectin-coated slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: hGAAP expression alters the number and size of focal adhesions. U2-OS cells transfected with siRNA (24 h) or plasmids encoding hGAAP were seeded onto fibronectin-coated slides. (A) Confocal images show cells transfected with vinculin-GFP (14 h), fixed, and stained with phalloidin–Alexa Fluor 568. (B) Summary results (means ± SEM from ≥25 cells for each condition) show numbers of focal adhesions per cell, determined by counting vinculin-GFP spots. (C) IRM images of U2-OS cells overexpressing hGAAP or hGAAP Ctmut, or transfected with the indicated siRNAs. Images are typical of three independent experiments. (D) Example of the image analysis used to determine focal adhesion number and size. Individual cells were imaged for IRM (a) and phalloidin staining (b). IRM images were then band pass filtered (c), and a threshold was imposed (d) to obtain the final images used to determine the number and size of the adhesions. (E and F) Summary results (means ± SEM from ≥20 cells for each condition) show numbers of adhesions per cell (E) and their areas (F). *, P

    Article Snippet: FACS analysis of cell surface integrins Cell surface expression of total and active β1 integrin in U2-OS cells seeded on 10 µg/ml fibronectin-coated dishes (Invitrogen) was determined by FACS (CyAn ADP multiple laser excitation; Dako).

    Techniques: Expressing, Transfection, Staining

    hGAAP stimulates calpain activity. (A) U2-OS cells were transfected with the CFP/YFP calpain FRET biosensor, plated on fibronectin, and imaged live over 20 min in both FRET and IRM channels. Typical examples show stills from videos. Right images show enlargements of boxed areas in left images. Bars, 5 µm. (B) Summary results from the images collected at t = 0 (means ± SEM, for > 10 cells) show the percentages of low-FRET pixels (ratio

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: hGAAP stimulates calpain activity. (A) U2-OS cells were transfected with the CFP/YFP calpain FRET biosensor, plated on fibronectin, and imaged live over 20 min in both FRET and IRM channels. Typical examples show stills from videos. Right images show enlargements of boxed areas in left images. Bars, 5 µm. (B) Summary results from the images collected at t = 0 (means ± SEM, for > 10 cells) show the percentages of low-FRET pixels (ratio

    Article Snippet: FACS analysis of cell surface integrins Cell surface expression of total and active β1 integrin in U2-OS cells seeded on 10 µg/ml fibronectin-coated dishes (Invitrogen) was determined by FACS (CyAn ADP multiple laser excitation; Dako).

    Techniques: Activity Assay, Transfection

    hGAAP expression alters focal adhesion dynamics. (A and E) U2-OS cells overexpressing hGAAP (A) or transfected with siRNAs (E) were transfected with vinculin-GFP and seeded onto fibronectin-coated dishes. After 30 min, individual cells were imaged at 2-min intervals for 2 h. Representative frames are shown, with arrows highlighting a single typical adhesion for each cell line. Bars, 10 µm. (B–D and F–H) Summary results (means ± SEM, n = 6 cells, with 4–10 focal adhesions analyzed in each) show mean lifetimes of focal adhesions (B and F) and rate constants for their disassembly (C and G) and assembly (D and H). **, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: hGAAP expression alters focal adhesion dynamics. (A and E) U2-OS cells overexpressing hGAAP (A) or transfected with siRNAs (E) were transfected with vinculin-GFP and seeded onto fibronectin-coated dishes. After 30 min, individual cells were imaged at 2-min intervals for 2 h. Representative frames are shown, with arrows highlighting a single typical adhesion for each cell line. Bars, 10 µm. (B–D and F–H) Summary results (means ± SEM, n = 6 cells, with 4–10 focal adhesions analyzed in each) show mean lifetimes of focal adhesions (B and F) and rate constants for their disassembly (C and G) and assembly (D and H). **, P

    Article Snippet: FACS analysis of cell surface integrins Cell surface expression of total and active β1 integrin in U2-OS cells seeded on 10 µg/ml fibronectin-coated dishes (Invitrogen) was determined by FACS (CyAn ADP multiple laser excitation; Dako).

    Techniques: Expressing, Transfection

    Overexpression of hGAAP increases the speed of cell migration. (A–F) U2-OS cells overexpressing hGAAP, hGAAP Ctmut, or control neo (A–C) or cells transfected with siRNA (D–F) were seeded at low density on fibronectin-coated dishes. Individual cells were imaged at 5-min intervals for 18 h. Tracks of individual cells ( n = 30) are shown in A and D. Migration rates (B and E) and persistence (where 1 represents a straight line of migration from start to finish; C and F) are shown as means ± SEM from 30 cells. **, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: Overexpression of hGAAP increases the speed of cell migration. (A–F) U2-OS cells overexpressing hGAAP, hGAAP Ctmut, or control neo (A–C) or cells transfected with siRNA (D–F) were seeded at low density on fibronectin-coated dishes. Individual cells were imaged at 5-min intervals for 18 h. Tracks of individual cells ( n = 30) are shown in A and D. Migration rates (B and E) and persistence (where 1 represents a straight line of migration from start to finish; C and F) are shown as means ± SEM from 30 cells. **, P

    Article Snippet: FACS analysis of cell surface integrins Cell surface expression of total and active β1 integrin in U2-OS cells seeded on 10 µg/ml fibronectin-coated dishes (Invitrogen) was determined by FACS (CyAn ADP multiple laser excitation; Dako).

    Techniques: Over Expression, Migration, Transfection

    Inhibition of calpain reduces the effects of hGAAP on cell migration and spreading. (A and B) Cells treated with PD150606 (A) or ALLM (B) were seeded on fibronectin-coated slides and left to adhere and spread for different times. Cells were fixed, and cell size was measured for > 50 cells per condition. (C–F) Cells treated with PD150606 (C and D) or ALLM (E and F) were seeded at low density in fibronectin-coated dishes, and cells were imaged every 5 min for 18 h. Individual cell tracks are shown in C and E ( n = 25 cells), and cumulative migration speeds from multiple migration tracks are shown in D and F. (G–J) Cells expressing hGAAP, hGAAP Ctmut, or control neo were treated with calpain 2–specific siRNAs. (G) IB showing effectiveness of calpain 2 knockdown in cells expressing hGAAP, hGAAP Ctmut, or control neo, and treated with calpain 2–specific siRNAs. (H) Areas of cells (measured 60 h after calpain 2 (Capn2) siRNA transfection, > 50 cells per condition) seeded on fibronectin-coated slides. (I and J) Cells treated with calpain 2 siRNA were seeded at low density in fibronectin-coated dishes, and individual cells were imaged every 5 min for 18 h. Individual cell tracks are shown ( n = 25 cells; I), and cumulative migration speeds are shown (J). Data are representative of three experiments and are shown as mean ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2

    doi: 10.1083/jcb.201301016

    Figure Lengend Snippet: Inhibition of calpain reduces the effects of hGAAP on cell migration and spreading. (A and B) Cells treated with PD150606 (A) or ALLM (B) were seeded on fibronectin-coated slides and left to adhere and spread for different times. Cells were fixed, and cell size was measured for > 50 cells per condition. (C–F) Cells treated with PD150606 (C and D) or ALLM (E and F) were seeded at low density in fibronectin-coated dishes, and cells were imaged every 5 min for 18 h. Individual cell tracks are shown in C and E ( n = 25 cells), and cumulative migration speeds from multiple migration tracks are shown in D and F. (G–J) Cells expressing hGAAP, hGAAP Ctmut, or control neo were treated with calpain 2–specific siRNAs. (G) IB showing effectiveness of calpain 2 knockdown in cells expressing hGAAP, hGAAP Ctmut, or control neo, and treated with calpain 2–specific siRNAs. (H) Areas of cells (measured 60 h after calpain 2 (Capn2) siRNA transfection, > 50 cells per condition) seeded on fibronectin-coated slides. (I and J) Cells treated with calpain 2 siRNA were seeded at low density in fibronectin-coated dishes, and individual cells were imaged every 5 min for 18 h. Individual cell tracks are shown ( n = 25 cells; I), and cumulative migration speeds are shown (J). Data are representative of three experiments and are shown as mean ± SEM. *, P

    Article Snippet: FACS analysis of cell surface integrins Cell surface expression of total and active β1 integrin in U2-OS cells seeded on 10 µg/ml fibronectin-coated dishes (Invitrogen) was determined by FACS (CyAn ADP multiple laser excitation; Dako).

    Techniques: Inhibition, Migration, Expressing, Transfection

    Flow chart for the protocol of simultaneous isolation of MSCs and EPCs derived from murine BM. BM cells were flushed out of femurs and tibias with DMEM complete medium. To obtain MSCs, the medium in culture dish A was gradually replaced with 1.5 ml fresh DMEM complete medium was performed every 8 h for 72 h. To obtain EPCs, non-adherent cells were plated in culture dish C, which was coated with human fibronectin, and maintained in endothelial growth medium. MSCs, mesenchymal stem or stromal cells; EPCs, endothelial progenitor cells; BM, bone marrow; DMEM, Dulbecco's modified Eagle's medium.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Simultaneous isolation of mesenchymal stem cells and endothelial progenitor cells derived from murine bone marrow

    doi: 10.3892/etm.2018.6844

    Figure Lengend Snippet: Flow chart for the protocol of simultaneous isolation of MSCs and EPCs derived from murine BM. BM cells were flushed out of femurs and tibias with DMEM complete medium. To obtain MSCs, the medium in culture dish A was gradually replaced with 1.5 ml fresh DMEM complete medium was performed every 8 h for 72 h. To obtain EPCs, non-adherent cells were plated in culture dish C, which was coated with human fibronectin, and maintained in endothelial growth medium. MSCs, mesenchymal stem or stromal cells; EPCs, endothelial progenitor cells; BM, bone marrow; DMEM, Dulbecco's modified Eagle's medium.

    Article Snippet: Then, non-adherent cells from 2 mice were collected, plated in a 60 mm culture dish coated with human fibronectin (Gibco; Thermo Fisher Scientific, Inc.) and maintained in endothelial growth medium (EGM), which contained endothelial cell ‘basal medium-2, EGM™ −2 MV SingleQuots™ (both Lonza Group, Ltd., Basel, Switzerland), 100 U/ml penicillin and 100 U/ml streptomycin ( ).

    Techniques: Flow Cytometry, Isolation, Derivative Assay, Modification

    Protein adsorption on SS-SPC. ( a ) Fluorescence microscopy images of Alexa Fluor® 488 labeled avidin and fibronectin adsorbed on unmodified SS (SS-ctrl) and SS-SPC in different conditions. The used protein concentration was either 3 μg/ml (C1) or 30 μg/ml (C2), and the exposure time 1 h (T1) or 3 h (T2). Scale bars 100 μm. ( b,c ) The boxplots show the mean fluorescence intensities of the adsorbed avidin and fibronectin, respectively, on SS-SPC (SPC) and unmodified SS (Ctrl) in the conditions depicted in ( a ).

    Journal: Scientific Reports

    Article Title: Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

    doi: 10.1038/srep29324

    Figure Lengend Snippet: Protein adsorption on SS-SPC. ( a ) Fluorescence microscopy images of Alexa Fluor® 488 labeled avidin and fibronectin adsorbed on unmodified SS (SS-ctrl) and SS-SPC in different conditions. The used protein concentration was either 3 μg/ml (C1) or 30 μg/ml (C2), and the exposure time 1 h (T1) or 3 h (T2). Scale bars 100 μm. ( b,c ) The boxplots show the mean fluorescence intensities of the adsorbed avidin and fibronectin, respectively, on SS-SPC (SPC) and unmodified SS (Ctrl) in the conditions depicted in ( a ).

    Article Snippet: Wildtype chicken avidin (Belovo, Belgium, MW = 16 kDa/monomer) and fibronectin (gelatin-affinity purified from human serum, MW = 220 kDa/monomer) had been previously labeled with Alexa Fluor® 488 NHS ester (Cat. No. A-20000, Thermo Fisher Scientific, Inc. Waltham, MA, USA) by Dr. J. Pärssinen according to manufacturer’s instructions.

    Techniques: Adsorption, Fluorescence, Microscopy, Labeling, Avidin-Biotin Assay, Protein Concentration