fibronectin  (Millipore)


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    Structured Review

    Millipore fibronectin
    <t>Fibronectin-</t> and LM05-F-conditioned media induce phosphorylation of ER-α at serine-118 in LM05-E cells. a Immunofluorescence analyzing the levels of phospho-serine 118 ER (pSer-118). LM05-Mix cells were grown to 80% confluency in growth medium
    Fibronectin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 362 article reviews
    Price from $9.99 to $1999.99
    fibronectin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through ?1 integrin"

    Article Title: The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through ?1 integrin

    Journal: Breast cancer research and treatment

    doi: 10.1007/s10549-011-1766-x

    Fibronectin- and LM05-F-conditioned media induce phosphorylation of ER-α at serine-118 in LM05-E cells. a Immunofluorescence analyzing the levels of phospho-serine 118 ER (pSer-118). LM05-Mix cells were grown to 80% confluency in growth medium
    Figure Legend Snippet: Fibronectin- and LM05-F-conditioned media induce phosphorylation of ER-α at serine-118 in LM05-E cells. a Immunofluorescence analyzing the levels of phospho-serine 118 ER (pSer-118). LM05-Mix cells were grown to 80% confluency in growth medium

    Techniques Used: Immunofluorescence

    Fibronectin (FN) induces tamoxifen resistance in LM05-E and MCF-7 cells. a Immunofluorescence for FN ( green, right panel ) in LM05-Mix cell cultures (nuclei were counterstained with DAPI, left panel ). FN was especially associated with the stromal compartment
    Figure Legend Snippet: Fibronectin (FN) induces tamoxifen resistance in LM05-E and MCF-7 cells. a Immunofluorescence for FN ( green, right panel ) in LM05-Mix cell cultures (nuclei were counterstained with DAPI, left panel ). FN was especially associated with the stromal compartment

    Techniques Used: Immunofluorescence

    Fibronectin induces phosphorylation of ER at serine-118 in MCF-7 cells through MAPK/ERK1/2. a MCF-7 cells were grown to 80% confluency and starved for 48 h in phenol red free DMEM/F12. They were then treated with vehicle (water) or FN (30 µg/ml)
    Figure Legend Snippet: Fibronectin induces phosphorylation of ER at serine-118 in MCF-7 cells through MAPK/ERK1/2. a MCF-7 cells were grown to 80% confluency and starved for 48 h in phenol red free DMEM/F12. They were then treated with vehicle (water) or FN (30 µg/ml)

    Techniques Used:

    2) Product Images from "Calcium-sensing Receptor Modulates Cell Adhesion and Migration via Integrins *"

    Article Title: Calcium-sensing Receptor Modulates Cell Adhesion and Migration via Integrins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.265454

    GRGDSP peptide inhibited NPS R-568-mediated cell adhesion. rMTC 44-2 cells were incubated on 1 μg/ml fibronectin or 2.5 μg/ml fibronectin in the presence of 300 n m R-568 with varying concentrations of the integrin-inhibiting peptide, GRGDSP,
    Figure Legend Snippet: GRGDSP peptide inhibited NPS R-568-mediated cell adhesion. rMTC 44-2 cells were incubated on 1 μg/ml fibronectin or 2.5 μg/ml fibronectin in the presence of 300 n m R-568 with varying concentrations of the integrin-inhibiting peptide, GRGDSP,

    Techniques Used: Incubation

    NPS R-568 increased fibronectin-mediated cell migration via integrin activation. rMTC 44-2 cells added to the upper surface of transwell filters migrated to the lower chamber coated with 10 μg/ml fibronectin. 1 μ m NPS R-568 induced a 2-fold
    Figure Legend Snippet: NPS R-568 increased fibronectin-mediated cell migration via integrin activation. rMTC 44-2 cells added to the upper surface of transwell filters migrated to the lower chamber coated with 10 μg/ml fibronectin. 1 μ m NPS R-568 induced a 2-fold

    Techniques Used: Migration, Activation Assay

    CaSR-positive allosteric modulator NPS R-568 potentiated fibronectin mediated cell adhesion. A , rMTC 44-2 cells were incubated on fibronectin-coated wells (0–15 μg/ml) in the absence or presence of 0.02% DMSO (control), 6 μ m NPS
    Figure Legend Snippet: CaSR-positive allosteric modulator NPS R-568 potentiated fibronectin mediated cell adhesion. A , rMTC 44-2 cells were incubated on fibronectin-coated wells (0–15 μg/ml) in the absence or presence of 0.02% DMSO (control), 6 μ m NPS

    Techniques Used: Incubation

    3) Product Images from "Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks"

    Article Title: Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00094

    Astrocytes isolated from primary rat cortex and cultured on paper-based substrates. (A–D) Influence of extracellular matrix protein coatings on astrocyte proliferation and viability at 7 DIV. Astrocytes were seeded at 100,000 cells/cm 2 on paper coated with (A) laminin, (B) fibronectin, or (C) Matrigel and on a control of (D) laminin-coated glass. After 7 days in culture with medium containing 10% FBS, the cells were stained with a live/dead viability assay. The cells on Matrigel-coated paper had the highest (E) viability and (F) number of adhered cells likely due to proliferation and/or adhesion properties in comparison to fibronectin and laminin coated paper. (G) Immunostaining with the astrocytic marker GFAP confirmed the astrocyte phenotype of the majority of rat cortex derived cells cultured for 14 days on Matrigel coated paper. Significance levels: * p
    Figure Legend Snippet: Astrocytes isolated from primary rat cortex and cultured on paper-based substrates. (A–D) Influence of extracellular matrix protein coatings on astrocyte proliferation and viability at 7 DIV. Astrocytes were seeded at 100,000 cells/cm 2 on paper coated with (A) laminin, (B) fibronectin, or (C) Matrigel and on a control of (D) laminin-coated glass. After 7 days in culture with medium containing 10% FBS, the cells were stained with a live/dead viability assay. The cells on Matrigel-coated paper had the highest (E) viability and (F) number of adhered cells likely due to proliferation and/or adhesion properties in comparison to fibronectin and laminin coated paper. (G) Immunostaining with the astrocytic marker GFAP confirmed the astrocyte phenotype of the majority of rat cortex derived cells cultured for 14 days on Matrigel coated paper. Significance levels: * p

    Techniques Used: Isolation, Cell Culture, Staining, Viability Assay, Immunostaining, Marker, Derivative Assay

    4) Product Images from "Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression"

    Article Title: Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression

    Journal: Nature Communications

    doi: 10.1038/ncomms14056

    Characterization of polyacrylamide (PAA) beads. ( a ) Bright-field image of polymerized PAA beads after filtration. Scale bar, 50 μm. ( b ) Fluorescence image of PAA microbeads containing trapped large polymers functionalized with FITC; scale bar, 50 μm. ( c ) Fluorescence image of coating of PAA beads with Cy3-Fibronectin; scale bar, 50 μm. ( d ) Distribution of size of polyacrylamide beads in dependance on the concentration of PFPE-PEG surfactant (1, 3 and 5%) during initial vortexing. ( e ) Characteristic diffusion time of SRB molecules, using FCS, within gels indicated uniformity of beads (small dispearsion) and mechanical properties. Small time of diffusion is characteristic for soft gels and increases with the volume fraction. Gels are defined by the acrylamide/bisacrylamide ratio. On the right, for 5/0.225 gels we show the effect of mixing on the uniformity of the batch. Each point corresponds to a single mesure of the diffusion time within the bead. ( f ) Uniformity and reproducibility is maintained between batches (sample 1, sample 2 and sample 3) of the same acrylamide/bisacrylamide ratio (5/0.225). ( e , f ) Mean±s.d.
    Figure Legend Snippet: Characterization of polyacrylamide (PAA) beads. ( a ) Bright-field image of polymerized PAA beads after filtration. Scale bar, 50 μm. ( b ) Fluorescence image of PAA microbeads containing trapped large polymers functionalized with FITC; scale bar, 50 μm. ( c ) Fluorescence image of coating of PAA beads with Cy3-Fibronectin; scale bar, 50 μm. ( d ) Distribution of size of polyacrylamide beads in dependance on the concentration of PFPE-PEG surfactant (1, 3 and 5%) during initial vortexing. ( e ) Characteristic diffusion time of SRB molecules, using FCS, within gels indicated uniformity of beads (small dispearsion) and mechanical properties. Small time of diffusion is characteristic for soft gels and increases with the volume fraction. Gels are defined by the acrylamide/bisacrylamide ratio. On the right, for 5/0.225 gels we show the effect of mixing on the uniformity of the batch. Each point corresponds to a single mesure of the diffusion time within the bead. ( f ) Uniformity and reproducibility is maintained between batches (sample 1, sample 2 and sample 3) of the same acrylamide/bisacrylamide ratio (5/0.225). ( e , f ) Mean±s.d.

    Techniques Used: Filtration, Fluorescence, Concentration Assay, Diffusion-based Assay, Sulforhodamine B Assay

    5) Product Images from "High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect"

    Article Title: High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

    Journal: eLife

    doi: 10.7554/eLife.38309

    IgSF-CAM-dependent neurite dynamic and axon orientation defects in Srd5a3 mutant cerebellum. ( A ) Neurite number per GC body cluster across 21 hr in laminin and poly-D-lysine-coated wells as measured with Incucyte live cell imaging system (Control, n = 3; En1-Cre; Srd5a3 fl/- , n = 3). ( B ) Representative image of control and En1-Cre; Srd5a3 fl/- GCs after 21 hr in laminin and poly-D-lysine-coated wells. Scale bar 100 μm. ( C ) Neurite number normalized to GC body clusters after 21 hr with laminin/poly-D-lysine coatings, fibronectin or IgSF-CAMs-coated wells (NrCAM and L1CAM). Each dot per coating represents results from a single mutant mouse. All GC cultures were performed in technical triplicates (Control, n = 3; En1-Cre; Srd5a3 fl/- , n = 3). ( D ) Neurite number and number of migrating neurons in GC re-aggregates plated for 24 hr on laminin and poly-D-lysine and ( E ) L1CAM coated surfaces. Five aggregates were quantified per mouse and coating condition (Control n = 5, En1Cre; Srd5a3 fl/- , n = 7). ( F ) Representative electron microscopy images of cerebellar ML sagittal view at P21 taken from control (n = 3) and Srd5a3 mutant (En1-Cre; Srd5a3 fl/- n = 1; Atoh1-Cre; Srd5a3 fl/- n = 2) mice. Scale bar 2 μm. Parallel fibers show a single orientation in control ML, whereas some exhibit an abnormal perpendicular orientation in the most outer ML in the mutant mice (G, red arrowhead). Two-tailed Student t-test was used for statistics. *p
    Figure Legend Snippet: IgSF-CAM-dependent neurite dynamic and axon orientation defects in Srd5a3 mutant cerebellum. ( A ) Neurite number per GC body cluster across 21 hr in laminin and poly-D-lysine-coated wells as measured with Incucyte live cell imaging system (Control, n = 3; En1-Cre; Srd5a3 fl/- , n = 3). ( B ) Representative image of control and En1-Cre; Srd5a3 fl/- GCs after 21 hr in laminin and poly-D-lysine-coated wells. Scale bar 100 μm. ( C ) Neurite number normalized to GC body clusters after 21 hr with laminin/poly-D-lysine coatings, fibronectin or IgSF-CAMs-coated wells (NrCAM and L1CAM). Each dot per coating represents results from a single mutant mouse. All GC cultures were performed in technical triplicates (Control, n = 3; En1-Cre; Srd5a3 fl/- , n = 3). ( D ) Neurite number and number of migrating neurons in GC re-aggregates plated for 24 hr on laminin and poly-D-lysine and ( E ) L1CAM coated surfaces. Five aggregates were quantified per mouse and coating condition (Control n = 5, En1Cre; Srd5a3 fl/- , n = 7). ( F ) Representative electron microscopy images of cerebellar ML sagittal view at P21 taken from control (n = 3) and Srd5a3 mutant (En1-Cre; Srd5a3 fl/- n = 1; Atoh1-Cre; Srd5a3 fl/- n = 2) mice. Scale bar 2 μm. Parallel fibers show a single orientation in control ML, whereas some exhibit an abnormal perpendicular orientation in the most outer ML in the mutant mice (G, red arrowhead). Two-tailed Student t-test was used for statistics. *p

    Techniques Used: Chick Chorioallantoic Membrane Assay, Mutagenesis, Live Cell Imaging, Electron Microscopy, Mouse Assay, Two Tailed Test

    6) Product Images from "High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect"

    Article Title: High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

    Journal: eLife

    doi: 10.7554/eLife.38309

    Changes in granule cell (GC) number and neurite length under different coating conditions. ( A ) GC body cluster number after 21 hr using different coatings and normalized to control littermates. ( B ) Neurite length in mutant GCs normalized to GC body cluster number after 21 hr. Each dot per coating represents results from a single mutant mouse. All GC cultures were performed in technical triplicates (n = 3 for each genotype). Student t-test was used for statistics. ( C ) Normalized neurite length of GCs under different coating conditions, laminin and polyD-lysine or fibronectin, and ( D ) L1CAM and NrCAM coating. *p
    Figure Legend Snippet: Changes in granule cell (GC) number and neurite length under different coating conditions. ( A ) GC body cluster number after 21 hr using different coatings and normalized to control littermates. ( B ) Neurite length in mutant GCs normalized to GC body cluster number after 21 hr. Each dot per coating represents results from a single mutant mouse. All GC cultures were performed in technical triplicates (n = 3 for each genotype). Student t-test was used for statistics. ( C ) Normalized neurite length of GCs under different coating conditions, laminin and polyD-lysine or fibronectin, and ( D ) L1CAM and NrCAM coating. *p

    Techniques Used: Mutagenesis

    7) Product Images from "The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells"

    Article Title: The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells

    Journal: Redox Biology

    doi: 10.1016/j.redox.2015.09.006

    H 2 O 2 consumption rates of HUVEC in different ECMs. Comparison of H 2 O 2 consumption rates (slope*(reaction volume)/(number of plated cells)) of HUVEC plated on dishes coated with gelatin, fibronectin or laminin, showed that consumption of H 2 O 2 by these cells was lower in the presence of laminin when compared with gelatin (values show the mean and SEM; N =3–8; Tukey test, ** p =0.005).
    Figure Legend Snippet: H 2 O 2 consumption rates of HUVEC in different ECMs. Comparison of H 2 O 2 consumption rates (slope*(reaction volume)/(number of plated cells)) of HUVEC plated on dishes coated with gelatin, fibronectin or laminin, showed that consumption of H 2 O 2 by these cells was lower in the presence of laminin when compared with gelatin (values show the mean and SEM; N =3–8; Tukey test, ** p =0.005).

    Techniques Used:

    8) Product Images from "Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression"

    Article Title: Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005324

    Snail is upregulated under extracellular matrix (ECM)-mediated signals. (A) Western blot analysis showing Snail expression on immobilized ECM. After HUVECs were transfected with siCon or siSnail, the transfectants were reseeded and cultured on PLL (20 μg/mL)-, FN (20 μg/mL)-, or CI (20 μg/mL)-coated culture dishes for 2 h. PLL, poly-L-lysine; FN, fibronectin; CI, collagen type I. (B) Time-course expression pattern of Snail on immobilized ECM. Confluent HUVECs were reseeded and cultured on PLL-, FN- or CI-coated dishes for the indicated time points. Snail expression was evaluated by western blot (upper) and quantitative RT-PCR (lower) analyses. (C) Western blot analysis showing the induction of phosphorylated Akt (p-Akt) and phosphorylated extracellular-regulated kinase 1/2 (p-Erk1/2) in HUVECs that were cultured on FN-coated dishes. (D) Snail expression on immobilized ECM after MK2206 treatment. Confluent HUVECs or human retinal endothelial cells (HRECs) were pre-exposed to 10 μM PP2 (a Src kinase inhibitor) or 1 μg/mL MK2206 (an allosteric Akt inhibitor) for 1 h, followed by reseeding and culture on PLL-, FN-, or CI-coated dishes for 2 h (western blot) or 1 h (quantitative RT-PCR).
    Figure Legend Snippet: Snail is upregulated under extracellular matrix (ECM)-mediated signals. (A) Western blot analysis showing Snail expression on immobilized ECM. After HUVECs were transfected with siCon or siSnail, the transfectants were reseeded and cultured on PLL (20 μg/mL)-, FN (20 μg/mL)-, or CI (20 μg/mL)-coated culture dishes for 2 h. PLL, poly-L-lysine; FN, fibronectin; CI, collagen type I. (B) Time-course expression pattern of Snail on immobilized ECM. Confluent HUVECs were reseeded and cultured on PLL-, FN- or CI-coated dishes for the indicated time points. Snail expression was evaluated by western blot (upper) and quantitative RT-PCR (lower) analyses. (C) Western blot analysis showing the induction of phosphorylated Akt (p-Akt) and phosphorylated extracellular-regulated kinase 1/2 (p-Erk1/2) in HUVECs that were cultured on FN-coated dishes. (D) Snail expression on immobilized ECM after MK2206 treatment. Confluent HUVECs or human retinal endothelial cells (HRECs) were pre-exposed to 10 μM PP2 (a Src kinase inhibitor) or 1 μg/mL MK2206 (an allosteric Akt inhibitor) for 1 h, followed by reseeding and culture on PLL-, FN-, or CI-coated dishes for 2 h (western blot) or 1 h (quantitative RT-PCR).

    Techniques Used: Western Blot, Expressing, Transfection, Cell Culture, Quantitative RT-PCR

    9) Product Images from "Gap geometry dictates epithelial closure efficiency"

    Article Title: Gap geometry dictates epithelial closure efficiency

    Journal: Nature Communications

    doi: 10.1038/ncomms8683

    Fibronectin concentration and pharmacological inhibition influence cell-crawling and purse-string contraction. Top row, area decay of the gap over time. The linear fitting of experimental data is shown by the dashed line. Insets, overlay impression of outlines at different time points from representative experiments. Bottom row, relation between velocity–curvature. Logarithmic (best fit for a ; R 2 =0.94-0.97) and linear fitting (best fit for b , c ) of experimental data are shown by dashed line as guide of the eyes. ( a ) Closure of gaps on substrates coated with 5, 20 and 40 μg ml −1 fibronectin ( n =15–19). Inset, representative experiment with 20 μg ml −1 fibronectin. ( b ) Blebbistatin inhibition of myosin II (50 μM) tested at 20 and 40 μg ml −1 of fibronectin ( n =9–19). Inset, myosin II inhibition at 40 μg ml −1 fibronectin. ( c ) CK666 inhibition of the ARP2/3 complex (10 and 100 μM) tested at 20 and 40 μg ml −1 fibronectin ( n =5–11). Inset, ARP2/3 inhibition at 10 μM CK666 and 20 μg ml −1 fibronectin. Error bars are s.e.m.
    Figure Legend Snippet: Fibronectin concentration and pharmacological inhibition influence cell-crawling and purse-string contraction. Top row, area decay of the gap over time. The linear fitting of experimental data is shown by the dashed line. Insets, overlay impression of outlines at different time points from representative experiments. Bottom row, relation between velocity–curvature. Logarithmic (best fit for a ; R 2 =0.94-0.97) and linear fitting (best fit for b , c ) of experimental data are shown by dashed line as guide of the eyes. ( a ) Closure of gaps on substrates coated with 5, 20 and 40 μg ml −1 fibronectin ( n =15–19). Inset, representative experiment with 20 μg ml −1 fibronectin. ( b ) Blebbistatin inhibition of myosin II (50 μM) tested at 20 and 40 μg ml −1 of fibronectin ( n =9–19). Inset, myosin II inhibition at 40 μg ml −1 fibronectin. ( c ) CK666 inhibition of the ARP2/3 complex (10 and 100 μM) tested at 20 and 40 μg ml −1 fibronectin ( n =5–11). Inset, ARP2/3 inhibition at 10 μM CK666 and 20 μg ml −1 fibronectin. Error bars are s.e.m.

    Techniques Used: Concentration Assay, Inhibition

    10) Product Images from "Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo"

    Article Title: Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo

    Journal: The FASEB Journal

    doi: 10.1096/fj.201700686RRR

    Human monocytes bind to Fbln7-FL and Fbln7-C via integrins and inhibit cell spreading. A ) Binding of unstimulated and TNF-α–stimulated monocytes to 96-well plates that were coated with fibronectin (5 µg/ml), Fbln7-FL (10 µg/ml), and Fbln7-C (10 µg/ml). Bar graph shows the quantification of relative binding compared with binding of unstimulated monocytes on a fibronectin substrate as 100. B – D ). Binding inhibition of TNF-α–stimulated monocytes plated on fibronectin ( B ), Fbln7-FL ( C ), and Fbln7-C ( D ) substrates by blocking Abs to integrins α v β 3 , α 2 β 1 , and α 5 β 1 . Cell binding activity on each substrate without the Ab was set as 100 and compared with cell binding activity with the Ab. Data represent the average of 3 independent experiments performed in triplicate. E ) Actin stress fiber formation of TNF-α–stimulated monocytes on fibronectin, Fbln7-FL, and Fbln7-C substrates by phalloidin staining. Images were taken with a ×63 objective. Scale bars, 5 µm. F ). Quantification of the cell area of the attached cells (100 cells) counted from the representative snapshots (×20) taken 30 min after the attachment of activated monocytes on each substrate by using ImageJ. ns, not significant. Data represent the results from 2 independent experiments. * P
    Figure Legend Snippet: Human monocytes bind to Fbln7-FL and Fbln7-C via integrins and inhibit cell spreading. A ) Binding of unstimulated and TNF-α–stimulated monocytes to 96-well plates that were coated with fibronectin (5 µg/ml), Fbln7-FL (10 µg/ml), and Fbln7-C (10 µg/ml). Bar graph shows the quantification of relative binding compared with binding of unstimulated monocytes on a fibronectin substrate as 100. B – D ). Binding inhibition of TNF-α–stimulated monocytes plated on fibronectin ( B ), Fbln7-FL ( C ), and Fbln7-C ( D ) substrates by blocking Abs to integrins α v β 3 , α 2 β 1 , and α 5 β 1 . Cell binding activity on each substrate without the Ab was set as 100 and compared with cell binding activity with the Ab. Data represent the average of 3 independent experiments performed in triplicate. E ) Actin stress fiber formation of TNF-α–stimulated monocytes on fibronectin, Fbln7-FL, and Fbln7-C substrates by phalloidin staining. Images were taken with a ×63 objective. Scale bars, 5 µm. F ). Quantification of the cell area of the attached cells (100 cells) counted from the representative snapshots (×20) taken 30 min after the attachment of activated monocytes on each substrate by using ImageJ. ns, not significant. Data represent the results from 2 independent experiments. * P

    Techniques Used: Binding Assay, Inhibition, Blocking Assay, Activity Assay, Staining

    11) Product Images from "Nerve Growth Factor Stimulates the Accumulation of β1 Integrin at the Tips of Filopodia in the Growth Cones of Sympathetic Neurons"

    Article Title: Nerve Growth Factor Stimulates the Accumulation of β1 Integrin at the Tips of Filopodia in the Growth Cones of Sympathetic Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.17-14-05455.1997

    ECM proteins are not necessary for the accumulation of β1 integrin at filopodial tips but cause aggregates of β1 integrin in growth cones previously treated with 100 ng/ml NGF to migrate rearward along filopodia. A , NGF causes the accumulation of β1 integrin tip aggregates (0–10 min) in the presence of 1 mg/ml RGDS peptide (•), much the same as NGF alone (○). Addition of 20 μg/ml fibronectin at 10 min ( arrowhead ) stimulates a rapid decrease in aggregates of β1 integrin at filopodial tips in the absence but not the presence of RGDS peptide. Results are representative of three separate experiments ( n = 200). B , Laminin 1 also causes a rapid delocalization of aggregates of β1 integrin from filopodial tips when added at a concentration of 20 μg/ml (indicated by the arrowhead ). Results are representative of three separate experiments ( n = 200). C , Typical growth cone treated with 100 ng/ml NGF for 10 min followed by 5 min of treatment with 20 μg/ml laminin 1. Aggregates of β1 integrin, visualized by immunofluorescence staining, can be seen situated along filopodial shafts ( arrow ). Scale bar, 10 μm. D , Laminin 1 causes rearward migration of aggregates of β1 integrin. Cells were treated as in C , and filopodia were scored for aggregates of β1 integrin at the tips and in the distal (excluding tips) and proximal halves of filopodia. Results are representative of three separate experiments ( n = 25). E , β1 Integrin aggregates are inserted in the membrane while at the tips of filopodia and during rearward migration. Cells were pretreated with NGF for 10 min, followed by 3 min of treatment with fibronectin, surface-stained for β1 integrin, and then scored as in D ( n = 25). In D and E , error bars show SDs. Asterisks denote those values that are significantly different ( p
    Figure Legend Snippet: ECM proteins are not necessary for the accumulation of β1 integrin at filopodial tips but cause aggregates of β1 integrin in growth cones previously treated with 100 ng/ml NGF to migrate rearward along filopodia. A , NGF causes the accumulation of β1 integrin tip aggregates (0–10 min) in the presence of 1 mg/ml RGDS peptide (•), much the same as NGF alone (○). Addition of 20 μg/ml fibronectin at 10 min ( arrowhead ) stimulates a rapid decrease in aggregates of β1 integrin at filopodial tips in the absence but not the presence of RGDS peptide. Results are representative of three separate experiments ( n = 200). B , Laminin 1 also causes a rapid delocalization of aggregates of β1 integrin from filopodial tips when added at a concentration of 20 μg/ml (indicated by the arrowhead ). Results are representative of three separate experiments ( n = 200). C , Typical growth cone treated with 100 ng/ml NGF for 10 min followed by 5 min of treatment with 20 μg/ml laminin 1. Aggregates of β1 integrin, visualized by immunofluorescence staining, can be seen situated along filopodial shafts ( arrow ). Scale bar, 10 μm. D , Laminin 1 causes rearward migration of aggregates of β1 integrin. Cells were treated as in C , and filopodia were scored for aggregates of β1 integrin at the tips and in the distal (excluding tips) and proximal halves of filopodia. Results are representative of three separate experiments ( n = 25). E , β1 Integrin aggregates are inserted in the membrane while at the tips of filopodia and during rearward migration. Cells were pretreated with NGF for 10 min, followed by 3 min of treatment with fibronectin, surface-stained for β1 integrin, and then scored as in D ( n = 25). In D and E , error bars show SDs. Asterisks denote those values that are significantly different ( p

    Techniques Used: Concentration Assay, Immunofluorescence, Staining, Migration

    12) Product Images from "Binding of Brucella protein, Bp26, to select extracellular matrix molecules"

    Article Title: Binding of Brucella protein, Bp26, to select extracellular matrix molecules

    Journal: BMC Molecular and Cell Biology

    doi: 10.1186/s12860-019-0239-7

    Binding of Bp26 protein to immobilized ECM components. Depiction of binding of collagen type I, fibronectin, vitronectin, laminin and bovine serum albumin (negative control) immobilized on a microtitre ELISA plate after overnight incubation at 4 °C with Bp26 protein (5 μg/ μl). Different concentrations of the molecules were detected by peroxidase reaction using anti-Bp26 mouse serum (diluted 1:1000) and rabbit anti-mouse IgG peroxidase conjugate and peroxidase substrate. The measures represent the average of each independent test after subtraction of the background value obtained in the absence of each the ECM molecules. Bars indicate standard errors presented as the means ± SD for each of the four tests
    Figure Legend Snippet: Binding of Bp26 protein to immobilized ECM components. Depiction of binding of collagen type I, fibronectin, vitronectin, laminin and bovine serum albumin (negative control) immobilized on a microtitre ELISA plate after overnight incubation at 4 °C with Bp26 protein (5 μg/ μl). Different concentrations of the molecules were detected by peroxidase reaction using anti-Bp26 mouse serum (diluted 1:1000) and rabbit anti-mouse IgG peroxidase conjugate and peroxidase substrate. The measures represent the average of each independent test after subtraction of the background value obtained in the absence of each the ECM molecules. Bars indicate standard errors presented as the means ± SD for each of the four tests

    Techniques Used: Binding Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Incubation

    Binding of Bp26 protein to soluble ECM components. Representation of different concentrations of collagen type I ( a ), fibronectin ( b ), and vitronectin ( c ) after incubation with immobilized Bp26 protein (5 μg/ μl) on ELISA microtiter plates and detection of binding affinity. Binding of the molecules was detected by peroxidase reaction using MABs specific for each molecule, followed by peroxidase conjugate and substrate as detailed in Materials Methods. The optical density values of the negative controls were subtracted from the binding values. Each value represents the mean ± SD for each of four independent tests. The ECM molecules concentrations are expressed in μg ml − 1
    Figure Legend Snippet: Binding of Bp26 protein to soluble ECM components. Representation of different concentrations of collagen type I ( a ), fibronectin ( b ), and vitronectin ( c ) after incubation with immobilized Bp26 protein (5 μg/ μl) on ELISA microtiter plates and detection of binding affinity. Binding of the molecules was detected by peroxidase reaction using MABs specific for each molecule, followed by peroxidase conjugate and substrate as detailed in Materials Methods. The optical density values of the negative controls were subtracted from the binding values. Each value represents the mean ± SD for each of four independent tests. The ECM molecules concentrations are expressed in μg ml − 1

    Techniques Used: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Biolayer interferometry analyses of Bp26 binding to ECM proteins. Heat-inactivated BSA ( a ), collagen I ( b ), fibronectin ( c ), and laminin ( d ) 521, 10 μg/ml in 10 mM sodium acetate, pH 4 (ForteBio), were respectively coupled onto AR2G sensors (ForteBio) with immobilization levels between 1.5 to 2.0 nm. For kinetics analysis, Bp26 was diluted in the running kinetics buffer (ForteBio) with additional 0.15 M NaCl to reduce the non-specific binding of Bp26 to the reference sensor. The concentrations tested were 0, 125, 250, 500, and 1000 nM. All the experiments were performed at 30 °C, including association for 5 min, and dissociation for 15 min. The raw data were processed by reference subtraction and data correction. E. Biolayer interferometry analyses of vitronectin binding to immobilized Bp26 Bionylated Bp26 was captured onto SA sensors (ForteBio) with immobilization levels of 2.0 nm. Vitronectin was diluted in the running kinetics buffer (ForteBio) to the concentrations of 75, 300, 600, and 1200 nM. All the experiments were performed at 30 °C, including association for 5 min, and dissociation for 15 min. The raw data were processed by reference subtraction and data correction.
    Figure Legend Snippet: Biolayer interferometry analyses of Bp26 binding to ECM proteins. Heat-inactivated BSA ( a ), collagen I ( b ), fibronectin ( c ), and laminin ( d ) 521, 10 μg/ml in 10 mM sodium acetate, pH 4 (ForteBio), were respectively coupled onto AR2G sensors (ForteBio) with immobilization levels between 1.5 to 2.0 nm. For kinetics analysis, Bp26 was diluted in the running kinetics buffer (ForteBio) with additional 0.15 M NaCl to reduce the non-specific binding of Bp26 to the reference sensor. The concentrations tested were 0, 125, 250, 500, and 1000 nM. All the experiments were performed at 30 °C, including association for 5 min, and dissociation for 15 min. The raw data were processed by reference subtraction and data correction. E. Biolayer interferometry analyses of vitronectin binding to immobilized Bp26 Bionylated Bp26 was captured onto SA sensors (ForteBio) with immobilization levels of 2.0 nm. Vitronectin was diluted in the running kinetics buffer (ForteBio) to the concentrations of 75, 300, 600, and 1200 nM. All the experiments were performed at 30 °C, including association for 5 min, and dissociation for 15 min. The raw data were processed by reference subtraction and data correction.

    Techniques Used: Binding Assay

    13) Product Images from "ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer"

    Article Title: ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24625

    ALCAM overexpression in mesenchymal Hec1A-ETV5 cells decreased cell migration and enhanced cell-cell adhesion (A) Representation of the two vectors constructed to overexpress ALCAM: full-length ALCAM and ALCAMcytoless inserted in the pmCherry-N1 construct. (B) Images of the Hec1A-ETV5 cell line transfected with the pmCherry vector as a control, and both ALCAM-overexpression constructs. (C) Western-blot analysis evidenced that ALCAM-Cherry was predominantly expressed in the cell membrane in Hec1A-ETV5 cells. (D) Images of the 3D spheroid-spreading model are illustrated at different time points. In the control cells, the spheroids were almost disaggregated after 5 h, whilst the mean time for disaggregation for the ALCAMcytoless cells was around 18 h, and more than 21 h for the full-length ALCAM cells. (E) Effect of ALCAM overexpression on migration was assessed by a 3D spheroid-spreading model on 200 μm fibronectin-coated stripes. On the left, box plot of the speed of migration for each cell line. On the right, representative images at time 0 h and at 24 h are illustrated and the spreading reached at 24 h is outlined in the images. Migration decreased in cells transfected with full-length ALCAM ( ** p
    Figure Legend Snippet: ALCAM overexpression in mesenchymal Hec1A-ETV5 cells decreased cell migration and enhanced cell-cell adhesion (A) Representation of the two vectors constructed to overexpress ALCAM: full-length ALCAM and ALCAMcytoless inserted in the pmCherry-N1 construct. (B) Images of the Hec1A-ETV5 cell line transfected with the pmCherry vector as a control, and both ALCAM-overexpression constructs. (C) Western-blot analysis evidenced that ALCAM-Cherry was predominantly expressed in the cell membrane in Hec1A-ETV5 cells. (D) Images of the 3D spheroid-spreading model are illustrated at different time points. In the control cells, the spheroids were almost disaggregated after 5 h, whilst the mean time for disaggregation for the ALCAMcytoless cells was around 18 h, and more than 21 h for the full-length ALCAM cells. (E) Effect of ALCAM overexpression on migration was assessed by a 3D spheroid-spreading model on 200 μm fibronectin-coated stripes. On the left, box plot of the speed of migration for each cell line. On the right, representative images at time 0 h and at 24 h are illustrated and the spreading reached at 24 h is outlined in the images. Migration decreased in cells transfected with full-length ALCAM ( ** p

    Techniques Used: Over Expression, Migration, Construct, Transfection, Plasmid Preparation, Western Blot

    14) Product Images from "Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein"

    Article Title: Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-8-14

    Fibronectin overlay assay performed on surface proteins of L. plantarum LM3 and LM3-CC1 . (A) PVDF membrane stained with Ponceau S. (B). Autoradiography of PVDF membrane after peroxidase assay. Lanes: 1, LM3; 2, LM3-CC1.
    Figure Legend Snippet: Fibronectin overlay assay performed on surface proteins of L. plantarum LM3 and LM3-CC1 . (A) PVDF membrane stained with Ponceau S. (B). Autoradiography of PVDF membrane after peroxidase assay. Lanes: 1, LM3; 2, LM3-CC1.

    Techniques Used: Overlay Assay, Staining, Autoradiography

    Binding of LM3 and LM3-CC1 to fibronectin immobilized on microtiter plate wells. Adhesion of both strains was evaluated by Real-time PCR . Adhesion is given as number of bacteria bound to fibronectin at different values of pH. Error bars represent ± standard deviation of the mean values.
    Figure Legend Snippet: Binding of LM3 and LM3-CC1 to fibronectin immobilized on microtiter plate wells. Adhesion of both strains was evaluated by Real-time PCR . Adhesion is given as number of bacteria bound to fibronectin at different values of pH. Error bars represent ± standard deviation of the mean values.

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    15) Product Images from "Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141"

    Article Title: Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004811

    US2-mediated downregulation of integrins inhibits downstream integrin-mediated signalling, cell adhesion and migration. ( A ) US2 inhibits signaling downstream of integrin α4β1. THP-1 cells were seeded on tissue culture plates coated +/- fibronectin as indicated for 1 hour at 37°C. Total cell lysates of adherent and non-adherent cells were analyzed by immunoblotting with phospho-specific antibody to tyr118 paxillin and total paxillin. ( B ) US2 inhibits THP-1 cell adhesion. THP-1 cells were seeded in triplicate on tissue culture plates coated with fibronectin as indicated for 1 hour at 37°C and washed 3 times with PBS. Adherent cells were quantified by CyQUANT-NF (Invitrogen). The percent adherent cells was normalized against control cells expressing empty vector (vector/US2 n = 3; US11/shbeta1 n = 2). ( C ) US2 inhibits cell migration. THP-1 cell migration +/- chemotaxis agent MCP-1 (10 nM) was examined in cells stably expressing US2, US11, control or ITGB1 shRNA (shbeta1) using fibronectin-coated Transwell plates. Number of cells migrated to the lower compartment is indicated (average of triplicates, n = 4). Data are represented as mean ± SEM. p-values were calculated using paired Student’s t-test.
    Figure Legend Snippet: US2-mediated downregulation of integrins inhibits downstream integrin-mediated signalling, cell adhesion and migration. ( A ) US2 inhibits signaling downstream of integrin α4β1. THP-1 cells were seeded on tissue culture plates coated +/- fibronectin as indicated for 1 hour at 37°C. Total cell lysates of adherent and non-adherent cells were analyzed by immunoblotting with phospho-specific antibody to tyr118 paxillin and total paxillin. ( B ) US2 inhibits THP-1 cell adhesion. THP-1 cells were seeded in triplicate on tissue culture plates coated with fibronectin as indicated for 1 hour at 37°C and washed 3 times with PBS. Adherent cells were quantified by CyQUANT-NF (Invitrogen). The percent adherent cells was normalized against control cells expressing empty vector (vector/US2 n = 3; US11/shbeta1 n = 2). ( C ) US2 inhibits cell migration. THP-1 cell migration +/- chemotaxis agent MCP-1 (10 nM) was examined in cells stably expressing US2, US11, control or ITGB1 shRNA (shbeta1) using fibronectin-coated Transwell plates. Number of cells migrated to the lower compartment is indicated (average of triplicates, n = 4). Data are represented as mean ± SEM. p-values were calculated using paired Student’s t-test.

    Techniques Used: Migration, CyQUANT Assay, Expressing, Plasmid Preparation, Chemotaxis Assay, Stable Transfection, shRNA

    US2/TRC8-mediated degradation of α integrins reduces cell adhesion of lytically HCMV infected THP-1 cells. ( A,B ) TRC8 depletion abolishes US2-induced α integrin degradation in HCMV infected THP-1 cells. THP-1 cells stably expressing a control shRNA (shCtrl) or shRNA targeting TRC8 (shTRC8) were PMA activated and infected with HCMV TB40 UL32-GFP (moi 25). Expression of indicated integrins was assessed at 96 hours post-infection by flow cytometry ( A ) or immunoblotting ( B ). ( C ) TRC8/US2-mediated degradation of α integrins reduces cell adhesion of HCMV-infected THP-1 cells. THP-1 cells were infected as above and at 96 hours post-infection counted, and seeded on tissue culture plates coated with fibronectin, recombinant VCAM-1-Fc, collagen or uncoated. After a 1 hour (fibronectin, VCAM-1, uncoated; n = 4) or 2 hours (collagen n = 2) incubation, loosely attached cells were washed off and the number of adherent cells was determined by CyQUANT-NF assay. Data show adhesion relative to fibronectin binding of control cells as mean ± SEM. p-values were calculated using paired Student’s t-test.
    Figure Legend Snippet: US2/TRC8-mediated degradation of α integrins reduces cell adhesion of lytically HCMV infected THP-1 cells. ( A,B ) TRC8 depletion abolishes US2-induced α integrin degradation in HCMV infected THP-1 cells. THP-1 cells stably expressing a control shRNA (shCtrl) or shRNA targeting TRC8 (shTRC8) were PMA activated and infected with HCMV TB40 UL32-GFP (moi 25). Expression of indicated integrins was assessed at 96 hours post-infection by flow cytometry ( A ) or immunoblotting ( B ). ( C ) TRC8/US2-mediated degradation of α integrins reduces cell adhesion of HCMV-infected THP-1 cells. THP-1 cells were infected as above and at 96 hours post-infection counted, and seeded on tissue culture plates coated with fibronectin, recombinant VCAM-1-Fc, collagen or uncoated. After a 1 hour (fibronectin, VCAM-1, uncoated; n = 4) or 2 hours (collagen n = 2) incubation, loosely attached cells were washed off and the number of adherent cells was determined by CyQUANT-NF assay. Data show adhesion relative to fibronectin binding of control cells as mean ± SEM. p-values were calculated using paired Student’s t-test.

    Techniques Used: Infection, Stable Transfection, Expressing, shRNA, Flow Cytometry, Cytometry, Recombinant, Incubation, CyQUANT Assay, Binding Assay

    16) Product Images from "Effect of alpha 2,6 Sialylation on Integrin-mediated Adhesion of Breast Cancer Cells to Fibronectin and Collagen IV"

    Article Title: Effect of alpha 2,6 Sialylation on Integrin-mediated Adhesion of Breast Cancer Cells to Fibronectin and Collagen IV

    Journal: Life sciences

    doi: 10.1016/j.lfs.2016.02.071

    Effect of sialidase on migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were grown on a fibronectin-coated plate to reach confluence and were treated with 0.1U/ml C. perfringens (CP), and A. ureafaciens (AU) sialidase in DPBS for 30 min
    Figure Legend Snippet: Effect of sialidase on migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were grown on a fibronectin-coated plate to reach confluence and were treated with 0.1U/ml C. perfringens (CP), and A. ureafaciens (AU) sialidase in DPBS for 30 min

    Techniques Used: Migration, Multiple Displacement Amplification

    17) Product Images from "Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality"

    Article Title: Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19010227

    Immunohistochemical analyses of native tissue and DT. Fibronectin, collagen IV and laminin were preserved in tissue decellularized with all protocols in all conditions. Scale bar = 70 µm.
    Figure Legend Snippet: Immunohistochemical analyses of native tissue and DT. Fibronectin, collagen IV and laminin were preserved in tissue decellularized with all protocols in all conditions. Scale bar = 70 µm.

    Techniques Used: Immunohistochemistry

    18) Product Images from "Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells"

    Article Title: Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-318

    Ectopic expression of Par3 protein enhances adhesiveness of the LNCaP cells . (A). 2 × 10 4 of LNCaP cells stably expressing Par3 protein or transfected with empty vector (EGFP) were seeded over fibronectin or collagen Type I pre-coated wells of a 96-well plate and incubated at 37°C for three hours, as described in the
    Figure Legend Snippet: Ectopic expression of Par3 protein enhances adhesiveness of the LNCaP cells . (A). 2 × 10 4 of LNCaP cells stably expressing Par3 protein or transfected with empty vector (EGFP) were seeded over fibronectin or collagen Type I pre-coated wells of a 96-well plate and incubated at 37°C for three hours, as described in the "Methods" section. After washing the wells with Dulbecco's phosphate-buffered saline the numbers of attached cells were calculated as the average from eight wells. The results were presented as the mean value ± S.D. The experiment was repeated three times with similar results. (B). Representative phase-contrast pictures (200×) of the cells attached to wells coated with fibronectin (FN) and collagen Type I (Col) after performing adhesion assay as described above for panel (A).

    Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Cell Adhesion Assay

    19) Product Images from "Dynamic inking of large-scale stamps for multiplexed microcontact printing and fabrication of cell microarrays"

    Article Title: Dynamic inking of large-scale stamps for multiplexed microcontact printing and fabrication of cell microarrays

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202531

    Cell microarray fabrication . (A) Representative scheme of cell microarray fabrication steps. (B) Fluorescence confocal images of PC3-GFP cells adhered on fibronectin patterned array. Nuclei were stained with DRAQ5™ dye (blue) and cytoplasms were expressing GFP (green). Dotted lines depict the edges of some printed features for clarity.
    Figure Legend Snippet: Cell microarray fabrication . (A) Representative scheme of cell microarray fabrication steps. (B) Fluorescence confocal images of PC3-GFP cells adhered on fibronectin patterned array. Nuclei were stained with DRAQ5™ dye (blue) and cytoplasms were expressing GFP (green). Dotted lines depict the edges of some printed features for clarity.

    Techniques Used: Microarray, Fluorescence, Staining, Expressing

    20) Product Images from "Focal adhesions undergo longitudinal splitting into fixed-width units"

    Article Title: Focal adhesions undergo longitudinal splitting into fixed-width units

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2018.04.073

    FA splitting in cells plated on fibronectin (A . (B) Fate of FAP during imaging period (00:37:00 to 01:14:30 h:min:sec). From the total number of FAP, graph shows percentage of FAP that do not split (example in A), split and create one or more stable FAU (split/remain), or split then completely disassemble (split/disassemble). N = 71 FAP, 6 cells. (C) Dot-plot of FAU widths from FN-plated U2OS live-cell videos such as those in A (mean 0.282 ± 0.04 μm, N = 136 FAU, 6 cells). (D) . (E) Fate of FAP during imaging period (00:52:00 to 01:29:30 h:min:sec). From the total number of FAP, graph shows percentage of FAP that do not split (example in D), split and create one or more stable FAU (split/remain), or completely disassemble (split/disassemble). (N = 27 FAP, 4 cells). (F) .
    Figure Legend Snippet: FA splitting in cells plated on fibronectin (A . (B) Fate of FAP during imaging period (00:37:00 to 01:14:30 h:min:sec). From the total number of FAP, graph shows percentage of FAP that do not split (example in A), split and create one or more stable FAU (split/remain), or split then completely disassemble (split/disassemble). N = 71 FAP, 6 cells. (C) Dot-plot of FAU widths from FN-plated U2OS live-cell videos such as those in A (mean 0.282 ± 0.04 μm, N = 136 FAU, 6 cells). (D) . (E) Fate of FAP during imaging period (00:52:00 to 01:29:30 h:min:sec). From the total number of FAP, graph shows percentage of FAP that do not split (example in D), split and create one or more stable FAU (split/remain), or completely disassemble (split/disassemble). (N = 27 FAP, 4 cells). (F) .

    Techniques Used: Imaging, Size-exclusion Chromatography

    21) Product Images from "Universally Conserved Relationships between Nuclear Shape and Cytoplasmic Mechanical Properties in Human Stem Cells"

    Article Title: Universally Conserved Relationships between Nuclear Shape and Cytoplasmic Mechanical Properties in Human Stem Cells

    Journal: Scientific Reports

    doi: 10.1038/srep23047

    Rheological effects of blocked F-actin polymerization on hASCs with micropatterned (μPP) morphology at subdiffusive loci (sDL) of the pnCSK. ( a ) Morphological constraint of hASCs via fibronectin-coated microablated PVA-film patterning (15 × 70 μPPs). ( b ) Significant changes on geometric and rheological parameters describing altered pnCSK mechanics in hASCs on μPP (15 μm × 70 μm rectangle) with or without 0.5 μM Cytochalasin D (CytoD) supplementation. ( c ) Discriminant analysis of control v. CytoD-induced cytoskeletal mechanics in hASCs with μPP-directed morphology. Error bars depict mean ± s.e.m interval (parameter effect significance: * p
    Figure Legend Snippet: Rheological effects of blocked F-actin polymerization on hASCs with micropatterned (μPP) morphology at subdiffusive loci (sDL) of the pnCSK. ( a ) Morphological constraint of hASCs via fibronectin-coated microablated PVA-film patterning (15 × 70 μPPs). ( b ) Significant changes on geometric and rheological parameters describing altered pnCSK mechanics in hASCs on μPP (15 μm × 70 μm rectangle) with or without 0.5 μM Cytochalasin D (CytoD) supplementation. ( c ) Discriminant analysis of control v. CytoD-induced cytoskeletal mechanics in hASCs with μPP-directed morphology. Error bars depict mean ± s.e.m interval (parameter effect significance: * p

    Techniques Used:

    A nucleus-centered elliptical coordinate system for perinuclear cytoskeleton (pnCSK) rheology. ( a ) Four-channel laser confocal microscopy of paraformaldehyde-fixed hASCs on fibronectin-coated coverslip cultures expressing an eGFP-actin fusion protein (green). Cells contain intracellular AlexaFluor 568-tagged beads (1-μm diameter, red) delivered by lipid-based endocytosis in culture. After fixation, cells were stained with Hoechst 33342 (blue) and phalloidin-AlexaFluor 633 (magenta) to highlight localization of nucleus and F-actin fibers, respectively. ( b ) Representative cell diagram depicting differences observed in dispersion of anisotropic mean squared displacements ≪Δr 2 (τ)≫ measured using either rectangular coordinates { x , y } or curvilinear nucleus-centered elliptical coordinates { u , v }. ( c ) Nondimensional parameterization of intracellular bead position in nucleus-centered elliptical coordinates. Bead coordinates are circularized relative to the nucleus of each cell by normalization to the locus u NE of its nuclear perimeter in elliptical coordinates; after normalization, nucleus-relative bead distances and angular pitch { Θ , Ω } are ensembled into a unified polar nondimensional map in which all nuclear perimeters trace along Θ = 1. ( d ) Parameterization of anisotropic intracellular rheology via a model power-law rheology model = ƒ n i for i = { u , v }. Complex shear moduli and are decomposed into their elastic and viscous terms G′ and G″ . In the frequency range 0.2 Hz
    Figure Legend Snippet: A nucleus-centered elliptical coordinate system for perinuclear cytoskeleton (pnCSK) rheology. ( a ) Four-channel laser confocal microscopy of paraformaldehyde-fixed hASCs on fibronectin-coated coverslip cultures expressing an eGFP-actin fusion protein (green). Cells contain intracellular AlexaFluor 568-tagged beads (1-μm diameter, red) delivered by lipid-based endocytosis in culture. After fixation, cells were stained with Hoechst 33342 (blue) and phalloidin-AlexaFluor 633 (magenta) to highlight localization of nucleus and F-actin fibers, respectively. ( b ) Representative cell diagram depicting differences observed in dispersion of anisotropic mean squared displacements ≪Δr 2 (τ)≫ measured using either rectangular coordinates { x , y } or curvilinear nucleus-centered elliptical coordinates { u , v }. ( c ) Nondimensional parameterization of intracellular bead position in nucleus-centered elliptical coordinates. Bead coordinates are circularized relative to the nucleus of each cell by normalization to the locus u NE of its nuclear perimeter in elliptical coordinates; after normalization, nucleus-relative bead distances and angular pitch { Θ , Ω } are ensembled into a unified polar nondimensional map in which all nuclear perimeters trace along Θ = 1. ( d ) Parameterization of anisotropic intracellular rheology via a model power-law rheology model = ƒ n i for i = { u , v }. Complex shear moduli and are decomposed into their elastic and viscous terms G′ and G″ . In the frequency range 0.2 Hz

    Techniques Used: Confocal Microscopy, Expressing, Staining

    22) Product Images from "Universally Conserved Relationships between Nuclear Shape and Cytoplasmic Mechanical Properties in Human Stem Cells"

    Article Title: Universally Conserved Relationships between Nuclear Shape and Cytoplasmic Mechanical Properties in Human Stem Cells

    Journal: Scientific Reports

    doi: 10.1038/srep23047

    Rheological effects of blocked F-actin polymerization on hASCs with micropatterned (μPP) morphology at subdiffusive loci (sDL) of the pnCSK. ( a ) Morphological constraint of hASCs via fibronectin-coated microablated PVA-film patterning (15 × 70 μPPs). ( b ) Significant changes on geometric and rheological parameters describing altered pnCSK mechanics in hASCs on μPP (15 μm × 70 μm rectangle) with or without 0.5 μM Cytochalasin D (CytoD) supplementation. ( c ) Discriminant analysis of control v. CytoD-induced cytoskeletal mechanics in hASCs with μPP-directed morphology. Error bars depict mean ± s.e.m interval (parameter effect significance: * p
    Figure Legend Snippet: Rheological effects of blocked F-actin polymerization on hASCs with micropatterned (μPP) morphology at subdiffusive loci (sDL) of the pnCSK. ( a ) Morphological constraint of hASCs via fibronectin-coated microablated PVA-film patterning (15 × 70 μPPs). ( b ) Significant changes on geometric and rheological parameters describing altered pnCSK mechanics in hASCs on μPP (15 μm × 70 μm rectangle) with or without 0.5 μM Cytochalasin D (CytoD) supplementation. ( c ) Discriminant analysis of control v. CytoD-induced cytoskeletal mechanics in hASCs with μPP-directed morphology. Error bars depict mean ± s.e.m interval (parameter effect significance: * p

    Techniques Used:

    A nucleus-centered elliptical coordinate system for perinuclear cytoskeleton (pnCSK) rheology. ( a ) Four-channel laser confocal microscopy of paraformaldehyde-fixed hASCs on fibronectin-coated coverslip cultures expressing an eGFP-actin fusion protein (green). Cells contain intracellular AlexaFluor 568-tagged beads (1-μm diameter, red) delivered by lipid-based endocytosis in culture. After fixation, cells were stained with Hoechst 33342 (blue) and phalloidin-AlexaFluor 633 (magenta) to highlight localization of nucleus and F-actin fibers, respectively. ( b ) Representative cell diagram depicting differences observed in dispersion of anisotropic mean squared displacements ≪Δr 2 (τ)≫ measured using either rectangular coordinates { x , y } or curvilinear nucleus-centered elliptical coordinates { u , v }. ( c ) Nondimensional parameterization of intracellular bead position in nucleus-centered elliptical coordinates. Bead coordinates are circularized relative to the nucleus of each cell by normalization to the locus u NE of its nuclear perimeter in elliptical coordinates; after normalization, nucleus-relative bead distances and angular pitch { Θ , Ω } are ensembled into a unified polar nondimensional map in which all nuclear perimeters trace along Θ = 1. ( d ) Parameterization of anisotropic intracellular rheology via a model power-law rheology model = ƒ n i for i = { u , v }. Complex shear moduli and are decomposed into their elastic and viscous terms G′ and G″ . In the frequency range 0.2 Hz
    Figure Legend Snippet: A nucleus-centered elliptical coordinate system for perinuclear cytoskeleton (pnCSK) rheology. ( a ) Four-channel laser confocal microscopy of paraformaldehyde-fixed hASCs on fibronectin-coated coverslip cultures expressing an eGFP-actin fusion protein (green). Cells contain intracellular AlexaFluor 568-tagged beads (1-μm diameter, red) delivered by lipid-based endocytosis in culture. After fixation, cells were stained with Hoechst 33342 (blue) and phalloidin-AlexaFluor 633 (magenta) to highlight localization of nucleus and F-actin fibers, respectively. ( b ) Representative cell diagram depicting differences observed in dispersion of anisotropic mean squared displacements ≪Δr 2 (τ)≫ measured using either rectangular coordinates { x , y } or curvilinear nucleus-centered elliptical coordinates { u , v }. ( c ) Nondimensional parameterization of intracellular bead position in nucleus-centered elliptical coordinates. Bead coordinates are circularized relative to the nucleus of each cell by normalization to the locus u NE of its nuclear perimeter in elliptical coordinates; after normalization, nucleus-relative bead distances and angular pitch { Θ , Ω } are ensembled into a unified polar nondimensional map in which all nuclear perimeters trace along Θ = 1. ( d ) Parameterization of anisotropic intracellular rheology via a model power-law rheology model = ƒ n i for i = { u , v }. Complex shear moduli and are decomposed into their elastic and viscous terms G′ and G″ . In the frequency range 0.2 Hz

    Techniques Used: Confocal Microscopy, Expressing, Staining

    23) Product Images from "Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish"

    Article Title: Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66584-8

    Appb regulates blastoderm cell adhesiveness. Embryos of wild-type or appb mutant background were injected with red (R) or green (G) fluorescent dextran at the one-cell stage, dissociated and mixed 1:1 at sphere stage and co-cultured in fibronectin coated plates ( A,B ). Cluster formation started around 6 hours post dissociation (hpd) ( C,D ) and was allowed to proceed for 16 hpd before image analysis ( E,F ). Scale bar = 200 μm. Frequency distributions of scattering ‘S’ ( G ) and dipole moment ‘P’ ( H ) of red and green cells in clusters of wt/wt (black line) and wt / appb mut (grey line). Dashed lines mark interval for scattering. *p
    Figure Legend Snippet: Appb regulates blastoderm cell adhesiveness. Embryos of wild-type or appb mutant background were injected with red (R) or green (G) fluorescent dextran at the one-cell stage, dissociated and mixed 1:1 at sphere stage and co-cultured in fibronectin coated plates ( A,B ). Cluster formation started around 6 hours post dissociation (hpd) ( C,D ) and was allowed to proceed for 16 hpd before image analysis ( E,F ). Scale bar = 200 μm. Frequency distributions of scattering ‘S’ ( G ) and dipole moment ‘P’ ( H ) of red and green cells in clusters of wt/wt (black line) and wt / appb mut (grey line). Dashed lines mark interval for scattering. *p

    Techniques Used: Mutagenesis, Injection, Cell Culture

    24) Product Images from "Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer"

    Article Title: Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer

    Journal: Scientific Reports

    doi: 10.1038/srep10194

    STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer. ( A ) Bar graph represents number of mammospheres formed from 2500 cells in presence and absence of indicated treatments. MDA-MB 231, MDA-MB 468 and MCF7 24 h mammosphere cultures were treated with Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). After 24 h, treatments were removed and cells were allowed to grow in fresh mammosphere culture media for 8 days. ( B ) Expression of various stem cell and EMT related transcription factors and markers were detected using western blotting in MDA-MB 231 cells with or without indicated treatments. The full size blots corresponding to the cropped blot images are given in Fig. S10 . ( C ) MDA-MB 231, MDA-MB 468 and MCF7 cells were pre-treated with either IL6 (100 ng ml −1 ), Fibronectin (1 μg ml −1 ) or EGF (25 ng ml −1 ) for two population doublings and subjected to mammosphere formation. Bar graph represents average of three independent experiments. ( D ) MCF7 cells were pre-treated with either IL6 (100 ng ml −1 ), Fibronectin (1 μg ml −1 ) or EGF (25 ng ml −1 ) for two population doublings and subjected to mammosphere formation. After 24 h, cells were treated with DMSO (untreated) or Shk (treated) as indicated in the bar graph. Data are shown as the mean ±SD. ( * ) p
    Figure Legend Snippet: STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer. ( A ) Bar graph represents number of mammospheres formed from 2500 cells in presence and absence of indicated treatments. MDA-MB 231, MDA-MB 468 and MCF7 24 h mammosphere cultures were treated with Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). After 24 h, treatments were removed and cells were allowed to grow in fresh mammosphere culture media for 8 days. ( B ) Expression of various stem cell and EMT related transcription factors and markers were detected using western blotting in MDA-MB 231 cells with or without indicated treatments. The full size blots corresponding to the cropped blot images are given in Fig. S10 . ( C ) MDA-MB 231, MDA-MB 468 and MCF7 cells were pre-treated with either IL6 (100 ng ml −1 ), Fibronectin (1 μg ml −1 ) or EGF (25 ng ml −1 ) for two population doublings and subjected to mammosphere formation. Bar graph represents average of three independent experiments. ( D ) MCF7 cells were pre-treated with either IL6 (100 ng ml −1 ), Fibronectin (1 μg ml −1 ) or EGF (25 ng ml −1 ) for two population doublings and subjected to mammosphere formation. After 24 h, cells were treated with DMSO (untreated) or Shk (treated) as indicated in the bar graph. Data are shown as the mean ±SD. ( * ) p

    Techniques Used: Activation Assay, Multiple Displacement Amplification, Expressing, Western Blot

    25) Product Images from "Resveratrol ameliorates high glucose-induced protein synthesis In glomerular epithelial cells"

    Article Title: Resveratrol ameliorates high glucose-induced protein synthesis In glomerular epithelial cells

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2009.09.011

    Resveratrol abolishes high glucose effects on protein synthesis and fibronectin expression in glomerular epithelial cells. A. Following 3-day exposure to 5 mM glucose (Con) or 30 mM glucose (Glc) with or without resveratrol (Res, 30 uM), de novo protein synthesis was measured by incorporation of 35 S-methionine (Met) into TCA-precipitable protein. Composite data from 3 experiments are shown in a graph (*p
    Figure Legend Snippet: Resveratrol abolishes high glucose effects on protein synthesis and fibronectin expression in glomerular epithelial cells. A. Following 3-day exposure to 5 mM glucose (Con) or 30 mM glucose (Glc) with or without resveratrol (Res, 30 uM), de novo protein synthesis was measured by incorporation of 35 S-methionine (Met) into TCA-precipitable protein. Composite data from 3 experiments are shown in a graph (*p

    Techniques Used: Expressing, Gas Chromatography

    26) Product Images from "Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts"

    Article Title: Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2015.10.027

    Mechanical stretch results in increased autophagy and decreased procollagen I and fibronectin in isolated cardiac fibroblasts
    Figure Legend Snippet: Mechanical stretch results in increased autophagy and decreased procollagen I and fibronectin in isolated cardiac fibroblasts

    Techniques Used: Isolation

    27) Product Images from "Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis"

    Article Title: Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2594

    QKI‐5 has a key role in endothelial cell (EC) differentiation derived from human induced pluripotent stem cells (hiPSCs). (A) : hiPSCs were generated and differentiated on low attachment plates using StemPro serum free media supplemented with BMP4, Activin A, fibroblast growth factor, and vascular endothelial growth factor (VEGF) for 5 days. KDR (VEGFR) positive population was selected using MicroBeads and cultured on Fibronectin coated plates supplemented with EGM‐2 media for 3–9 days. iPS‐ECs‐derived cells expressed a typical pattern for EC specific markers CD144, CD31, eNOS, and vWF as immunofluorescence images shown. (B) : FACs analysis showing cells positive for EC specific marker CD144 (C, D) QKI‐5 was expressed during EC differentiation in parallel with VE‐cadherin expression in mRNA level. (E–G) QKI‐5 was overexpressed by lentiviral gene transfer in hiPSCs on day 3 after KDR selection and the cells were harvesting on day 6 of EC differentiation showing a significant induction of the EC markers CD144, CD31, and eNOS, and signaling of VEGFR (KDR), VEGFA, and STAT3 as Western blots and real time data shown. (H, I) Luciferase assays shown that QKI‐5 induced the transcriptional activation of CD144 and VEGFR. (J) : Overexpression of QKI‐5 directs differentiation toward arterial EC as cells express specific marker Ephrin B2 and not venous (CoupTFII) or lymphatic (Lyve1) EC markers (data are means ± SEM [ n = 3]; *, p
    Figure Legend Snippet: QKI‐5 has a key role in endothelial cell (EC) differentiation derived from human induced pluripotent stem cells (hiPSCs). (A) : hiPSCs were generated and differentiated on low attachment plates using StemPro serum free media supplemented with BMP4, Activin A, fibroblast growth factor, and vascular endothelial growth factor (VEGF) for 5 days. KDR (VEGFR) positive population was selected using MicroBeads and cultured on Fibronectin coated plates supplemented with EGM‐2 media for 3–9 days. iPS‐ECs‐derived cells expressed a typical pattern for EC specific markers CD144, CD31, eNOS, and vWF as immunofluorescence images shown. (B) : FACs analysis showing cells positive for EC specific marker CD144 (C, D) QKI‐5 was expressed during EC differentiation in parallel with VE‐cadherin expression in mRNA level. (E–G) QKI‐5 was overexpressed by lentiviral gene transfer in hiPSCs on day 3 after KDR selection and the cells were harvesting on day 6 of EC differentiation showing a significant induction of the EC markers CD144, CD31, and eNOS, and signaling of VEGFR (KDR), VEGFA, and STAT3 as Western blots and real time data shown. (H, I) Luciferase assays shown that QKI‐5 induced the transcriptional activation of CD144 and VEGFR. (J) : Overexpression of QKI‐5 directs differentiation toward arterial EC as cells express specific marker Ephrin B2 and not venous (CoupTFII) or lymphatic (Lyve1) EC markers (data are means ± SEM [ n = 3]; *, p

    Techniques Used: Derivative Assay, Generated, Cell Culture, Immunofluorescence, FACS, Marker, Expressing, Selection, Western Blot, Luciferase, Activation Assay, Over Expression

    28) Product Images from "Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2"

    Article Title: Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

    Journal: Molecular and Cellular Biology

    doi:

    Pyk2 adhesion-dependent tyrosine phosphorylation is impaired by FAK expression. (A) Relative Pyk2 levels were determined by immunoblot (IB) analysis of total cell lysates (30 μg of total protein) from FAK-null cells (parental population to Tet-FAK clones) versus normal mouse embryo fibroblasts. (B) Induced or noninduced Tet-FAK(F397)-21 cells were serum starved for 2 h and then lysed when either attached (Att.), trypsinized and held in suspension for 30 min (Susp.), or replated onto fibronectin for either 30 min (Fn-30′) or 60 min (Fn-60′). Either FAK or Pyk2 was immunoprecipitated (IP) from 300 μg of total protein lysates and then divided equally for immunoblot (IB) detection of either FAK, Pyk2, or phosphotyrosine (pTyr).
    Figure Legend Snippet: Pyk2 adhesion-dependent tyrosine phosphorylation is impaired by FAK expression. (A) Relative Pyk2 levels were determined by immunoblot (IB) analysis of total cell lysates (30 μg of total protein) from FAK-null cells (parental population to Tet-FAK clones) versus normal mouse embryo fibroblasts. (B) Induced or noninduced Tet-FAK(F397)-21 cells were serum starved for 2 h and then lysed when either attached (Att.), trypsinized and held in suspension for 30 min (Susp.), or replated onto fibronectin for either 30 min (Fn-30′) or 60 min (Fn-60′). Either FAK or Pyk2 was immunoprecipitated (IP) from 300 μg of total protein lysates and then divided equally for immunoblot (IB) detection of either FAK, Pyk2, or phosphotyrosine (pTyr).

    Techniques Used: Expressing, Immunoprecipitation

    Induced FAK expression alters Cas phosphotyrosine levels. Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-29 cells were either maintained in the presence of tetracycline or induced to express near-equal amounts of WT-, F397, and F576/F577-FAK protein, respectively, and then cells from either attached (Att) (serum starved for 14 h), suspended (Sus), or fibronectin-replated (Fn) (30 min) conditions were lysed in RIPA buffer. FAK (top panel) and Cas (bottom panel) levels were assessed by immunoblot (IB) analysis of 20 μg of total protein from the lysates. Two hundred eighty micrograms of total protein from the same lysates was subjected to immunoprecipitation (IP) by using PY20 antiphosphotyrosine (pTyr) antibody, followed by Cas immunoblot analysis, to determine relative levels of tyrosine phosphorylation of Cas (middle panel).
    Figure Legend Snippet: Induced FAK expression alters Cas phosphotyrosine levels. Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-29 cells were either maintained in the presence of tetracycline or induced to express near-equal amounts of WT-, F397, and F576/F577-FAK protein, respectively, and then cells from either attached (Att) (serum starved for 14 h), suspended (Sus), or fibronectin-replated (Fn) (30 min) conditions were lysed in RIPA buffer. FAK (top panel) and Cas (bottom panel) levels were assessed by immunoblot (IB) analysis of 20 μg of total protein from the lysates. Two hundred eighty micrograms of total protein from the same lysates was subjected to immunoprecipitation (IP) by using PY20 antiphosphotyrosine (pTyr) antibody, followed by Cas immunoblot analysis, to determine relative levels of tyrosine phosphorylation of Cas (middle panel).

    Techniques Used: Expressing, Immunoprecipitation

    Late spreading analysis of Tet-FAK cells. Induced and noninduced Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-16 cells were plated onto fibronectin-coated glass coverslips and, after 20 h, phase-contrast light microscopic images were captured for spreading analysis. Note the highly spread morphology of induced F397-FAK cells. Bar = 75 μm.
    Figure Legend Snippet: Late spreading analysis of Tet-FAK cells. Induced and noninduced Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-16 cells were plated onto fibronectin-coated glass coverslips and, after 20 h, phase-contrast light microscopic images were captured for spreading analysis. Note the highly spread morphology of induced F397-FAK cells. Bar = 75 μm.

    Techniques Used:

    Immunolocalization of FAK in induced Tet-FAK cells. Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-16 cells were induced for 2 days and then plated overnight on fibronectin-coated coverslips. Double-label indirect immunofluorescence was carried out by using monoclonal antibody 8d4 against talin (left panels) and polyclonal antibody C-20 against FAK (right panels). Bar = 30 μm.
    Figure Legend Snippet: Immunolocalization of FAK in induced Tet-FAK cells. Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-16 cells were induced for 2 days and then plated overnight on fibronectin-coated coverslips. Double-label indirect immunofluorescence was carried out by using monoclonal antibody 8d4 against talin (left panels) and polyclonal antibody C-20 against FAK (right panels). Bar = 30 μm.

    Techniques Used: Immunofluorescence

    In vitro kinase assays. (A) Inhibition of c-Src but not FAK by PD161430. Increasing concentrations of PD161430 (0 to 220 μM) were added to mixtures for kinase reactions carried out on either coimmunoprecipitates of c-Src and p120 ctn (top panel) or baculovirus-expressed FAK (bottom panel). PD161430 inhibited c-Src phosphorylation of p120 ctn with a 50% inhibitory concentration of ∼0.2 μM but had little or no effect on FAK autophosphorylation at concentrations up to 220 μM. (B and C) F576/F577-FAK shows reduced adhesion-dependent tyrosine phosphorylation and in vitro autophosphorylation activity. WT-FAK or F576/F577-FAK was immunoprecipitated (IP) from NP-40 buffer lysates of induced Tet-FAK(WT)-46 or Tet-FAK(F576/F577)-16 cells, respectively, under either attached (Att) (serum-starved 14 h), suspended (Sus), or fibronectin-replated (Fn) conditions. The immunoprecipitates were then divided equally for assessment of in vivo FAK tyrosine phosphorylation by immunoblotting (IB) with antiphosphotyrosine antibody 4G10 (top panel), FAK recovery by immunoblotting with anti-FAK antibody C-20 (middle panel), and in vitro FAK phosphorylation from kinase assays carried out either in the absence or presence of 22 μM PD161430 (bottom panel). In panel C, in vitro FAK phosphorylation from three independent kinase assays is plotted as mean activity (+SEM) relative to that of WT-FAK assayed from attached cells in the absence of PD161430. (D) F397-FAK and F576/F577-FAK show reduced phosphorylation of poly(GluTyr). WT, -F397, and F576/F577-FAK were immunoprecipitated from NP-40 buffer lysates prepared from induced Tet-FAK(WT)-46, Tet-FAK(F397)-21, or Tet-FAK(F576/F577)-16 cells, respectively, and used to assess vitro kinase phosphorylation of poly(GluTyr) in kinase assays carried out either in the absence or presence of 22 μM PD161430. Data from three independent assays are plotted as mean activities (+SEM) relative to that of WT-FAK assayed from attached (Att) cells in the absence of PD161430.
    Figure Legend Snippet: In vitro kinase assays. (A) Inhibition of c-Src but not FAK by PD161430. Increasing concentrations of PD161430 (0 to 220 μM) were added to mixtures for kinase reactions carried out on either coimmunoprecipitates of c-Src and p120 ctn (top panel) or baculovirus-expressed FAK (bottom panel). PD161430 inhibited c-Src phosphorylation of p120 ctn with a 50% inhibitory concentration of ∼0.2 μM but had little or no effect on FAK autophosphorylation at concentrations up to 220 μM. (B and C) F576/F577-FAK shows reduced adhesion-dependent tyrosine phosphorylation and in vitro autophosphorylation activity. WT-FAK or F576/F577-FAK was immunoprecipitated (IP) from NP-40 buffer lysates of induced Tet-FAK(WT)-46 or Tet-FAK(F576/F577)-16 cells, respectively, under either attached (Att) (serum-starved 14 h), suspended (Sus), or fibronectin-replated (Fn) conditions. The immunoprecipitates were then divided equally for assessment of in vivo FAK tyrosine phosphorylation by immunoblotting (IB) with antiphosphotyrosine antibody 4G10 (top panel), FAK recovery by immunoblotting with anti-FAK antibody C-20 (middle panel), and in vitro FAK phosphorylation from kinase assays carried out either in the absence or presence of 22 μM PD161430 (bottom panel). In panel C, in vitro FAK phosphorylation from three independent kinase assays is plotted as mean activity (+SEM) relative to that of WT-FAK assayed from attached cells in the absence of PD161430. (D) F397-FAK and F576/F577-FAK show reduced phosphorylation of poly(GluTyr). WT, -F397, and F576/F577-FAK were immunoprecipitated from NP-40 buffer lysates prepared from induced Tet-FAK(WT)-46, Tet-FAK(F397)-21, or Tet-FAK(F576/F577)-16 cells, respectively, and used to assess vitro kinase phosphorylation of poly(GluTyr) in kinase assays carried out either in the absence or presence of 22 μM PD161430. Data from three independent assays are plotted as mean activities (+SEM) relative to that of WT-FAK assayed from attached (Att) cells in the absence of PD161430.

    Techniques Used: In Vitro, Inhibition, Concentration Assay, Activity Assay, Immunoprecipitation, In Vivo

    Early spreading analysis of Tet-FAK cells. Induced or noninduced Tet-FAK(WT)-46, Tet-FAK(F397)-21, or Tet-FAK(F576/F577)-16 cells were plated onto fibronectin-coated tissue culture dishes and, after 30 min, phase-contrast light microscopic images were captured for spreading analysis. Note full lamellipod extension of induced WT-FAK cells and more apparent filopod extension of induced F397-FAK cells. Bar = 37.5 μm.
    Figure Legend Snippet: Early spreading analysis of Tet-FAK cells. Induced or noninduced Tet-FAK(WT)-46, Tet-FAK(F397)-21, or Tet-FAK(F576/F577)-16 cells were plated onto fibronectin-coated tissue culture dishes and, after 30 min, phase-contrast light microscopic images were captured for spreading analysis. Note full lamellipod extension of induced WT-FAK cells and more apparent filopod extension of induced F397-FAK cells. Bar = 37.5 μm.

    Techniques Used:

    29) Product Images from "Indirect three‐dimensional printing: A method for fabricating polyurethane‐urea based cardiac scaffolds"

    Article Title: Indirect three‐dimensional printing: A method for fabricating polyurethane‐urea based cardiac scaffolds

    Journal: Journal of Biomedical Materials Research. Part a

    doi: 10.1002/jbm.a.35721

    The viability of cardiac myocytes after 24 h on fibronectin coated and uncoated PUU scaffolds, and 2D tissue culture plastic controls was tested using the PrestoBlue assay ( n = 3). Statistically significant differences ( p
    Figure Legend Snippet: The viability of cardiac myocytes after 24 h on fibronectin coated and uncoated PUU scaffolds, and 2D tissue culture plastic controls was tested using the PrestoBlue assay ( n = 3). Statistically significant differences ( p

    Techniques Used: Prestoblue Assay

    30) Product Images from "A Comparative Characterization of Different Host-sourced Lactobacillus ruminis Strains and Their Adhesive, Inhibitory, and Immunomodulating Functions"

    Article Title: A Comparative Characterization of Different Host-sourced Lactobacillus ruminis Strains and Their Adhesive, Inhibitory, and Immunomodulating Functions

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00657

    Inhibition of pathogen adherence to ECM proteins cells by L. ruminis . The inhibition of F4-fimbriated ETEC adherence to type I collagen (A,C,E) and the inhibition of Yersinia enterocolitica DSM 13030 T adherence to fibronectin and type I collagen (B,D,F) by L. ruminis strains in competition (A,B) , exclusion (C,D) , and displacement (E,F) assays was tested with 3 H-labeled ETEC and Yersinia cells as detailed in Section “Materials and Methods.” The means and standard deviations of three independent experiments are shown, each with three technical replicates.
    Figure Legend Snippet: Inhibition of pathogen adherence to ECM proteins cells by L. ruminis . The inhibition of F4-fimbriated ETEC adherence to type I collagen (A,C,E) and the inhibition of Yersinia enterocolitica DSM 13030 T adherence to fibronectin and type I collagen (B,D,F) by L. ruminis strains in competition (A,B) , exclusion (C,D) , and displacement (E,F) assays was tested with 3 H-labeled ETEC and Yersinia cells as detailed in Section “Materials and Methods.” The means and standard deviations of three independent experiments are shown, each with three technical replicates.

    Techniques Used: Inhibition, Labeling

    31) Product Images from "IL-4 impairs wound healing potential in the skin by repressing fibronectin expression"

    Article Title: IL-4 impairs wound healing potential in the skin by repressing fibronectin expression

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2016.07.012

    IL-4-stimulated keratinocytes show delayed wound healing ( A ) Human keratinocytes (HK) were differentiated for 5 days with calcium chloride in the presence or absence of IL-4 before analysis of FN1 expression by qRT-PCR. (B) Cell lysates were collected and the expression of fibronectin and β-actin was determined by western blot ( C ) Gene expression was analyzed 3h after scratching using qRT–PCR. ( D ) Percentage of scratch closed after 24h of culture, and ( E ) microscopy of human keratinocytes differentiated with calcium chloride alone or calcium chloride plus IL-4 for 5 days in control or plates covered with a substratum of BSA or fibronectin. Dashed red lines represent original wounds. Pictures are representative of 4 fields analyzed over at least three independent experiments. Data represents the mean of three independent experiments. *p
    Figure Legend Snippet: IL-4-stimulated keratinocytes show delayed wound healing ( A ) Human keratinocytes (HK) were differentiated for 5 days with calcium chloride in the presence or absence of IL-4 before analysis of FN1 expression by qRT-PCR. (B) Cell lysates were collected and the expression of fibronectin and β-actin was determined by western blot ( C ) Gene expression was analyzed 3h after scratching using qRT–PCR. ( D ) Percentage of scratch closed after 24h of culture, and ( E ) microscopy of human keratinocytes differentiated with calcium chloride alone or calcium chloride plus IL-4 for 5 days in control or plates covered with a substratum of BSA or fibronectin. Dashed red lines represent original wounds. Pictures are representative of 4 fields analyzed over at least three independent experiments. Data represents the mean of three independent experiments. *p

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

    32) Product Images from "CP30, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytoadherence"

    Article Title: CP30, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytoadherence

    Journal: Infection and Immunity

    doi:

    CP30 degrades different substrates at specific pHs. The CP30 proteolytic activity was analyzed on 0.2% gelatin (Gel); 800 μg of collagen IV (Coll IV); and 500 μg of fibronectin (Fn). The proteolytic activation was performed at 37°C at different pHs, as follows: lane 1, 3.6; lane 2, 4.5; lane 3, 5.0; lane 4, 5.5; lane 5, 6.0; lane 6, 6.5; and lane 7, 7.0. pH 4.5 was used as a control (lane 2).
    Figure Legend Snippet: CP30 degrades different substrates at specific pHs. The CP30 proteolytic activity was analyzed on 0.2% gelatin (Gel); 800 μg of collagen IV (Coll IV); and 500 μg of fibronectin (Fn). The proteolytic activation was performed at 37°C at different pHs, as follows: lane 1, 3.6; lane 2, 4.5; lane 3, 5.0; lane 4, 5.5; lane 5, 6.0; lane 6, 6.5; and lane 7, 7.0. pH 4.5 was used as a control (lane 2).

    Techniques Used: Activity Assay, Activation Assay

    CP30 is secreted (A) and is selective for different human protein substrates (B). (A) In vitro secretion of CP30 from parasites grown in suspension was compared to trichomonads grown in coculture over a HeLa cell monolayer (2 × 10 6 ). The same amounts of parasites (10 × 10 6 ) were inoculated in 30 ml of culture media for both conditions and incubated at 37°C for 18 h to produce 40 × 10 6 parasites. After parasites were pelleted by centrifugation at 900 × g , 1-ml aliquots from different sample supernatants (Media) were processed for a ligand assay (see Materials and Methods). Lanes: 1, samples from control fresh media; 2, spent culture media from parasites grown in suspension; 3, spent culture media from parasites grown in coculture with fixed HeLa cell monolayers; 4 and 5, clarified supernatant (1 ml) from parasites after 30 min in suspension (control [lane 4]) or in interaction with HeLa cell monolayers (lane 5) (adherence assay) which also were processed for a ligand assay; 6 through 8, material eluted from the same amount (10 6 ) of fixed HeLa cells was loaded from a mock control without contact with parasite lysates (lane 6) or in contact with extracts from parasites grown in suspension (lane 7) or from monolayers obtained from coculture with T. vaginalis (lane 8). All samples were analyzed by substrate gelatin gel electrophoresis. The positions of the molecular size markers are on the left. (B) The CP30 proteolytic activity was tested by SDS-PAGE using different human proteins as substrates. Lanes: 1, 0.2% gelatin (Gel); 2, 800 μg of collagen IV (Coll IV); 3, 500 μg of fibronectin (Fn); 4, 500 μg of laminin-1 (Lam-1); 5, 0.2% hemoglobin (Hb). All substrate gels were activated under the same experimental conditions, 24 h at 37°C and pH 5.0.
    Figure Legend Snippet: CP30 is secreted (A) and is selective for different human protein substrates (B). (A) In vitro secretion of CP30 from parasites grown in suspension was compared to trichomonads grown in coculture over a HeLa cell monolayer (2 × 10 6 ). The same amounts of parasites (10 × 10 6 ) were inoculated in 30 ml of culture media for both conditions and incubated at 37°C for 18 h to produce 40 × 10 6 parasites. After parasites were pelleted by centrifugation at 900 × g , 1-ml aliquots from different sample supernatants (Media) were processed for a ligand assay (see Materials and Methods). Lanes: 1, samples from control fresh media; 2, spent culture media from parasites grown in suspension; 3, spent culture media from parasites grown in coculture with fixed HeLa cell monolayers; 4 and 5, clarified supernatant (1 ml) from parasites after 30 min in suspension (control [lane 4]) or in interaction with HeLa cell monolayers (lane 5) (adherence assay) which also were processed for a ligand assay; 6 through 8, material eluted from the same amount (10 6 ) of fixed HeLa cells was loaded from a mock control without contact with parasite lysates (lane 6) or in contact with extracts from parasites grown in suspension (lane 7) or from monolayers obtained from coculture with T. vaginalis (lane 8). All samples were analyzed by substrate gelatin gel electrophoresis. The positions of the molecular size markers are on the left. (B) The CP30 proteolytic activity was tested by SDS-PAGE using different human proteins as substrates. Lanes: 1, 0.2% gelatin (Gel); 2, 800 μg of collagen IV (Coll IV); 3, 500 μg of fibronectin (Fn); 4, 500 μg of laminin-1 (Lam-1); 5, 0.2% hemoglobin (Hb). All substrate gels were activated under the same experimental conditions, 24 h at 37°C and pH 5.0.

    Techniques Used: In Vitro, Incubation, Centrifugation, Nucleic Acid Electrophoresis, Activity Assay, SDS Page, Laser Capture Microdissection

    33) Product Images from "Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation"

    Article Title: Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation

    Journal:

    doi: 10.1038/labinvest.2008.89

    D283 medulloblastoma cells are more adherent to H4 matrix than tissue culture plastic, laminin, or fibronectin. Phase-contrast images demonstrate the minimal adhesion characteristic of D283 cells grown on tissue culture plastic ( a ); the degree adhesion
    Figure Legend Snippet: D283 medulloblastoma cells are more adherent to H4 matrix than tissue culture plastic, laminin, or fibronectin. Phase-contrast images demonstrate the minimal adhesion characteristic of D283 cells grown on tissue culture plastic ( a ); the degree adhesion

    Techniques Used:

    34) Product Images from "Human NK Cells Lyse Th2-Polarizing Dendritic Cells via NKp30 and DNAM-1"

    Article Title: Human NK Cells Lyse Th2-Polarizing Dendritic Cells via NKp30 and DNAM-1

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1800475

    DCs treated with SEA show distinct morphology and spreading behavior. ( A ) Fluorescent images of immature DCs or DCs treated for 24 h with LPS or SEA, incubated on 10 μg/ml fibronectin, and stained with AF488-labeled phalloidin (to mark F-actin). Representative from 50 images taken with three independent donors; scale bar, 20 μm. ( B ) Spreading area of DCs measured from images as in (A) pooled from three donors. ( C ) Time-lapse interference reflection microscopy (IRM) images of DCs spreading on 10 μg/ml fibronectin over 15 min. Images show differently silhouettes of the adherent membrane color coded by time. Data are representative of 8–10 images obtained with three independent donors. ( D ) Total distance traveled by immature DCs or DCs treated for 24 h with LPS or SEA, plated on 10 μg/ml fibronectin, and tracked by time-lapse imaging over 5 h. ( E ) Average migration speed of immature DCs or DCs treated for 24 h with LPS or SEA, plated on 10 μg/ml fibronectin, and tracked by time-lapse imaging over 5 h. ( F ) Mean squared displacement (MSD) diffusion coefficient values calculated from multiple tracks of immature DCs or DCs treated for 24 h with LPS or SEA and plated on 10 μg/ml fibronectin for 5 h. In (B), (D), and (E), circles represent data points from individual cells; lines show mean (±SD) of cells pooled from three independent donors. In (F), circles represent MSD of imaging regions from three independent experiments; bars show mean (±SD) analyzed by one-way ANOVA with Tukey multiple comparisons (no significant differences). ** p
    Figure Legend Snippet: DCs treated with SEA show distinct morphology and spreading behavior. ( A ) Fluorescent images of immature DCs or DCs treated for 24 h with LPS or SEA, incubated on 10 μg/ml fibronectin, and stained with AF488-labeled phalloidin (to mark F-actin). Representative from 50 images taken with three independent donors; scale bar, 20 μm. ( B ) Spreading area of DCs measured from images as in (A) pooled from three donors. ( C ) Time-lapse interference reflection microscopy (IRM) images of DCs spreading on 10 μg/ml fibronectin over 15 min. Images show differently silhouettes of the adherent membrane color coded by time. Data are representative of 8–10 images obtained with three independent donors. ( D ) Total distance traveled by immature DCs or DCs treated for 24 h with LPS or SEA, plated on 10 μg/ml fibronectin, and tracked by time-lapse imaging over 5 h. ( E ) Average migration speed of immature DCs or DCs treated for 24 h with LPS or SEA, plated on 10 μg/ml fibronectin, and tracked by time-lapse imaging over 5 h. ( F ) Mean squared displacement (MSD) diffusion coefficient values calculated from multiple tracks of immature DCs or DCs treated for 24 h with LPS or SEA and plated on 10 μg/ml fibronectin for 5 h. In (B), (D), and (E), circles represent data points from individual cells; lines show mean (±SD) of cells pooled from three independent donors. In (F), circles represent MSD of imaging regions from three independent experiments; bars show mean (±SD) analyzed by one-way ANOVA with Tukey multiple comparisons (no significant differences). ** p

    Techniques Used: Incubation, Staining, Labeling, Microscopy, Imaging, Migration, Diffusion-based Assay

    35) Product Images from "Development and characterisation of a large diameter decellularised vascular allograft"

    Article Title: Development and characterisation of a large diameter decellularised vascular allograft

    Journal: Cell and Tissue Banking

    doi: 10.1007/s10561-017-9673-y

    Sections of ( a , c , e , g , i , k ) native and ( b , d , f , h , j , l ) acellular human aorta labelled using monoclonal antibodies against ( a , b ) Collagen type I ( c , d ) Collagen type III ( e , f ) Collagen type IV ( g , h ) Von Willebrand factor, ( i , j ) Fibronectin and ( k , l ) Laminin. Images were acquired using Kohler illumination and a × 10 objective, scale bars represent 200 µm
    Figure Legend Snippet: Sections of ( a , c , e , g , i , k ) native and ( b , d , f , h , j , l ) acellular human aorta labelled using monoclonal antibodies against ( a , b ) Collagen type I ( c , d ) Collagen type III ( e , f ) Collagen type IV ( g , h ) Von Willebrand factor, ( i , j ) Fibronectin and ( k , l ) Laminin. Images were acquired using Kohler illumination and a × 10 objective, scale bars represent 200 µm

    Techniques Used:

    36) Product Images from "Counting growth factors in single cells with infrared quantum dots to measure discrete stimulation distributions"

    Article Title: Counting growth factors in single cells with infrared quantum dots to measure discrete stimulation distributions

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08754-5

    Single-cell epidermal growth factor (EGF) binding correlates with single-cell receptor translocation and drug response. a Representative three-dimensional (3D) images of MDA-MB-231 cells after stimulation with quantum dot-EGF (QD-EGF) in the absence or presence of EGFR inhibitor gefitinib. Times after the start of a stimulation pulse are indicated. QDs are shown in red, nuclei are blue, and Alexa Fluor 488-conjugated fibronectin micropatterns are green. b Two-dimensional (2D) z -projections on xy fibronectin micropattern planes and c one-dimensional (1D) projections on x -axes indicate the localization of single EGF averaged across cells. d Representative image of cell membrane measured through fluorescence imaging of fluorescently labeled receptors (top) and membrane reconstruction using alpha shapes (bottom). e Correlation between EGF number and fraction of EGF internalized in individual cells at 10 and 30 min after the start of a QD-EGF stimulation pulse. f Western blots and g relative pEGFR abundance in MDA-MB-231 whole-cell lysates immediately after stimulation with QD-EGF in the presence of indicated gefitinib concentrations. Uncropped western blots with molecular weight markers are shown in Supplementary Figure 16 . h Fraction of EGF internalized in single cells at different gefitinib concentrations, 30 min after the start of a QD-EGF pulse. The box indicates 25/75th percentile; red lines are means; whiskers are s.d. i Coefficient of variation (CV) of the fraction of EGF internalized in h . j Number of EGF bound impacts the fraction of EGF internalized 10 min after the start of a QD-EGF pulse in the presence of gefitinib at 0, 51, and 5,100 nM concentration. The gray line shown in 51 nM (middle) and 5100 nM (right) gefitinib plots is the linear fit for 0 nM gefitinib condition (left). Data fits are shown in Supplementary Figure 14 . N = 20 and 12 cells for 10 and 30 min after QD-EGF stimulation onset without gefitinib, respectively; N = 12, 10, 20, 23, 12, and 14 cells for 30 min after QD-EGF stimulation onset in the presence of gefitinib at 0, 0.51, 5.1, 51, 510, and 5100 nM concentrations, respectively. All stimulation pulses used 1 nM EGF-QD for 5 min. All scale bars indicate 10 µm
    Figure Legend Snippet: Single-cell epidermal growth factor (EGF) binding correlates with single-cell receptor translocation and drug response. a Representative three-dimensional (3D) images of MDA-MB-231 cells after stimulation with quantum dot-EGF (QD-EGF) in the absence or presence of EGFR inhibitor gefitinib. Times after the start of a stimulation pulse are indicated. QDs are shown in red, nuclei are blue, and Alexa Fluor 488-conjugated fibronectin micropatterns are green. b Two-dimensional (2D) z -projections on xy fibronectin micropattern planes and c one-dimensional (1D) projections on x -axes indicate the localization of single EGF averaged across cells. d Representative image of cell membrane measured through fluorescence imaging of fluorescently labeled receptors (top) and membrane reconstruction using alpha shapes (bottom). e Correlation between EGF number and fraction of EGF internalized in individual cells at 10 and 30 min after the start of a QD-EGF stimulation pulse. f Western blots and g relative pEGFR abundance in MDA-MB-231 whole-cell lysates immediately after stimulation with QD-EGF in the presence of indicated gefitinib concentrations. Uncropped western blots with molecular weight markers are shown in Supplementary Figure 16 . h Fraction of EGF internalized in single cells at different gefitinib concentrations, 30 min after the start of a QD-EGF pulse. The box indicates 25/75th percentile; red lines are means; whiskers are s.d. i Coefficient of variation (CV) of the fraction of EGF internalized in h . j Number of EGF bound impacts the fraction of EGF internalized 10 min after the start of a QD-EGF pulse in the presence of gefitinib at 0, 51, and 5,100 nM concentration. The gray line shown in 51 nM (middle) and 5100 nM (right) gefitinib plots is the linear fit for 0 nM gefitinib condition (left). Data fits are shown in Supplementary Figure 14 . N = 20 and 12 cells for 10 and 30 min after QD-EGF stimulation onset without gefitinib, respectively; N = 12, 10, 20, 23, 12, and 14 cells for 30 min after QD-EGF stimulation onset in the presence of gefitinib at 0, 0.51, 5.1, 51, 510, and 5100 nM concentrations, respectively. All stimulation pulses used 1 nM EGF-QD for 5 min. All scale bars indicate 10 µm

    Techniques Used: Binding Assay, Translocation Assay, Multiple Displacement Amplification, Fluorescence, Imaging, Labeling, Western Blot, Molecular Weight, Concentration Assay

    Quantum dot (QD) calibrated three-dimensional (3D) deconvolution microscopy (QDC-3DM). a Schematic representation of the contribution of single-cell stimulation distribution (growth factor binding) to signaling response distribution (measured by receptor internalization). b Depiction of the QDC-3DM image analysis methodology to count growth factors in single cells. The process begins with acquisition of 3D fluorescence images of single cells to localize single QDs and spatially register their locations. A representative 3D image shows a cell stimulated with QD-epidermal growth factor (QD-EGF) (red) on an Alexa Fluor 488-labeled fibronectin substrate (green) with nucleus labeled with Hoechst (blue). Each 3D image is deconvolved and spatially correlated to two-dimensional (2D) videos in the QD color channel. In the first step shown at right, time traces of spot intensities are used to identify single QDs by their distinctive two-component intensity distributions. In the second step, the average intensity of these single QDs from 3D deconvolved images, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {I_{1{\mathrm{QD}}}^{3{\mathrm{DD}}}}$$\end{document} I 1 QD 3 DD ¯ , is measured. In the third step, the 3D intensity of each spot, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$I_{{\mathrm{spot}}}^{3{\mathrm{DD}}}$$\end{document} I spot 3 DD , is measured and registered to the average single-QD intensity, to calculate the number of QD-EGF per spot, N QD,spot . The number of EGF per cell, N EGF,cell , is then calculated as the sum of all N QD,spot
    Figure Legend Snippet: Quantum dot (QD) calibrated three-dimensional (3D) deconvolution microscopy (QDC-3DM). a Schematic representation of the contribution of single-cell stimulation distribution (growth factor binding) to signaling response distribution (measured by receptor internalization). b Depiction of the QDC-3DM image analysis methodology to count growth factors in single cells. The process begins with acquisition of 3D fluorescence images of single cells to localize single QDs and spatially register their locations. A representative 3D image shows a cell stimulated with QD-epidermal growth factor (QD-EGF) (red) on an Alexa Fluor 488-labeled fibronectin substrate (green) with nucleus labeled with Hoechst (blue). Each 3D image is deconvolved and spatially correlated to two-dimensional (2D) videos in the QD color channel. In the first step shown at right, time traces of spot intensities are used to identify single QDs by their distinctive two-component intensity distributions. In the second step, the average intensity of these single QDs from 3D deconvolved images, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline {I_{1{\mathrm{QD}}}^{3{\mathrm{DD}}}}$$\end{document} I 1 QD 3 DD ¯ , is measured. In the third step, the 3D intensity of each spot, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$I_{{\mathrm{spot}}}^{3{\mathrm{DD}}}$$\end{document} I spot 3 DD , is measured and registered to the average single-QD intensity, to calculate the number of QD-EGF per spot, N QD,spot . The number of EGF per cell, N EGF,cell , is then calculated as the sum of all N QD,spot

    Techniques Used: Microscopy, Cell Stimulation, Binding Assay, Fluorescence, Labeling

    37) Product Images from "A Biosynthetic Nerve Guide Conduit Based on Silk/SWNT/Fibronectin Nanocomposite for Peripheral Nerve Regeneration"

    Article Title: A Biosynthetic Nerve Guide Conduit Based on Silk/SWNT/Fibronectin Nanocomposite for Peripheral Nerve Regeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074417

    Scanning electron micrographs of: a) porous structure of freeze-died SF/SWNT conduits, b) Aligned fibronectin nanofibers produced through electrospining process.
    Figure Legend Snippet: Scanning electron micrographs of: a) porous structure of freeze-died SF/SWNT conduits, b) Aligned fibronectin nanofibers produced through electrospining process.

    Techniques Used: Produced

    Fibronectin bioactivity after 14 days in vitro study. FN exhibited high bioactivity (about 85%).
    Figure Legend Snippet: Fibronectin bioactivity after 14 days in vitro study. FN exhibited high bioactivity (about 85%).

    Techniques Used: In Vitro

    38) Product Images from "Heat shock protein 90 is an essential molecular chaperone for CB2 cannabinoid receptor-mediated signaling in trabecular meshwork cells"

    Article Title: Heat shock protein 90 is an essential molecular chaperone for CB2 cannabinoid receptor-mediated signaling in trabecular meshwork cells

    Journal: Molecular Vision

    doi:

    The effects of PD98059 and geldanamycin on CB2 receptor-mediated actin cytoskeleton changes in trabecular meshwork cells. Intact trabecular meshwork (TM) cells were plated on fibronectin-coated (5 μg/ml) coverslips and grown to confluence. Serum-deprived TM cells were pretreated with vehicle ( A and B ), 30 μM PD98059 ( C and D ) for 30 min and 10 μM geldanamycin (GA; E and F ) for 2 h. Subsequently, the TM cells were treated for 3 h with vehicle ( A ), 100 nM JWH015 ( B ), 30 μM PD98059 ( C ), 100 nM JWH015 plus 30 μM PD98059 ( D ), 10 μM geldanamycin ( E ), and 100 nM JWH015 plus 10 μM geldanamycin ( F ). The TM cells were then fixed with paraformaldehyde and stained with Alexafluor 488-labeled phalloidin as detailed in the Methods section. JWH015 caused a significant decrease in staining for actin stress fibers, compared with vehicle control. PD98059 as well as geldanamycin pretreatment blocked the changes brought about by JWH015.
    Figure Legend Snippet: The effects of PD98059 and geldanamycin on CB2 receptor-mediated actin cytoskeleton changes in trabecular meshwork cells. Intact trabecular meshwork (TM) cells were plated on fibronectin-coated (5 μg/ml) coverslips and grown to confluence. Serum-deprived TM cells were pretreated with vehicle ( A and B ), 30 μM PD98059 ( C and D ) for 30 min and 10 μM geldanamycin (GA; E and F ) for 2 h. Subsequently, the TM cells were treated for 3 h with vehicle ( A ), 100 nM JWH015 ( B ), 30 μM PD98059 ( C ), 100 nM JWH015 plus 30 μM PD98059 ( D ), 10 μM geldanamycin ( E ), and 100 nM JWH015 plus 10 μM geldanamycin ( F ). The TM cells were then fixed with paraformaldehyde and stained with Alexafluor 488-labeled phalloidin as detailed in the Methods section. JWH015 caused a significant decrease in staining for actin stress fibers, compared with vehicle control. PD98059 as well as geldanamycin pretreatment blocked the changes brought about by JWH015.

    Techniques Used: Staining, Labeling

    39) Product Images from "Identification and Clonal Characterisation of a Progenitor Cell Sub-Population in Normal Human Articular Cartilage"

    Article Title: Identification and Clonal Characterisation of a Progenitor Cell Sub-Population in Normal Human Articular Cartilage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013246

    Population data from clonal cell lines. Colony forming efficiency calculated from 8 samples that were subjected to the fibronectin adhesion assay (as described in Materials Methods ) (A). Population doublings data from 2 representative samples demonstrating proliferative rate of the clonal cell lines during a period of over 200 days (B). Phase contrast microscopic appearance of a representative fibronectin adhered colony (C) and clonal monolayers at low (
    Figure Legend Snippet: Population data from clonal cell lines. Colony forming efficiency calculated from 8 samples that were subjected to the fibronectin adhesion assay (as described in Materials Methods ) (A). Population doublings data from 2 representative samples demonstrating proliferative rate of the clonal cell lines during a period of over 200 days (B). Phase contrast microscopic appearance of a representative fibronectin adhered colony (C) and clonal monolayers at low (

    Techniques Used: Cell Adhesion Assay

    40) Product Images from "Notch Pathway Modulation on Bone Marrow-Derived Vascular Precursor Cells Regulates Their Angiogenic and Wound Healing Potential"

    Article Title: Notch Pathway Modulation on Bone Marrow-Derived Vascular Precursor Cells Regulates Their Angiogenic and Wound Healing Potential

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003752

    Notch pathway early inhibition impairs BM-PC adhesion and spreading to extracellular matrix, reducing the number of mature cells obtained at the end of the differentiation. A. Expression of Hes 1 and Hey 2 72 h after GSI (10 uM) treatment was detected by RT-PCR. B. Quantification of adherent BM-PC 72 h after treatment with DMSO, GSI at 10 ηM or 10 µM, on 2% gelatin coated wells. C. Quantification of adherent BM-PC 48 h after treatment with DMSO and GSI at 10 µM, on 2% gelatin, fibronectin, collagen or laminin coated wells. D. Quantification of control or GSI BM-PC expressing double EC – lineage specific markers (acLDL/FLK-1 or acLDL/VWF) after 20 days of endothelial differentiation. E. Representative image (100×) of adherent cells under the different conditions. *P
    Figure Legend Snippet: Notch pathway early inhibition impairs BM-PC adhesion and spreading to extracellular matrix, reducing the number of mature cells obtained at the end of the differentiation. A. Expression of Hes 1 and Hey 2 72 h after GSI (10 uM) treatment was detected by RT-PCR. B. Quantification of adherent BM-PC 72 h after treatment with DMSO, GSI at 10 ηM or 10 µM, on 2% gelatin coated wells. C. Quantification of adherent BM-PC 48 h after treatment with DMSO and GSI at 10 µM, on 2% gelatin, fibronectin, collagen or laminin coated wells. D. Quantification of control or GSI BM-PC expressing double EC – lineage specific markers (acLDL/FLK-1 or acLDL/VWF) after 20 days of endothelial differentiation. E. Representative image (100×) of adherent cells under the different conditions. *P

    Techniques Used: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

    Related Articles

    Flow Cytometry:

    Article Title: Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks
    Article Snippet: .. After activation, the papers were transferred into a sterile 24-well plate under the laminar flow hood and immersed in one of the following surface functionalizing solutions: 10 μg/mL of laminin (L-2020, Sigma-Aldrich) in Neurobasal medium, 20 μg/mL of fibronectin (F1141, Sigma-Aldrich) in Neurobasal medium, or 200 μL of Matrigel (Corning Matrigel Basement Membrane Matrix; 354230, Chemie Brunschwig AG, Switzerland) diluted to 2 mg/mL in ice cold serum-free medium. .. After 45 min of incubation at room temperature, the papers were rinsed with supplemented Neurobasal medium and kept in Neurobasal medium and 2% penicillin streptomycin until seeding.

    Article Title: Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells
    Article Snippet: .. To facilitate ECM protein coatings, collagen type I (from rat tail, cat.# 1179179, Roche Applied Science, Indianapolis, IN), collagen type IV (from human placenta, cat.# C7521, Sigma Aldrich, St. Louis, MO), and fibronectin (from bovine plasma, cat.# F4759, Sigma Aldrich) were prepared as sterile aqueous solutions (0.1 mg/ml) and independently incubated with silk scaffolds for 1 h at 37°C followed by 1 h of drying in a laminar flow hood at room temperature. .. Uncoated controls were prepared in parallel with incubation in PBS.

    Activation Assay:

    Article Title: Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks
    Article Snippet: .. After activation, the papers were transferred into a sterile 24-well plate under the laminar flow hood and immersed in one of the following surface functionalizing solutions: 10 μg/mL of laminin (L-2020, Sigma-Aldrich) in Neurobasal medium, 20 μg/mL of fibronectin (F1141, Sigma-Aldrich) in Neurobasal medium, or 200 μL of Matrigel (Corning Matrigel Basement Membrane Matrix; 354230, Chemie Brunschwig AG, Switzerland) diluted to 2 mg/mL in ice cold serum-free medium. .. After 45 min of incubation at room temperature, the papers were rinsed with supplemented Neurobasal medium and kept in Neurobasal medium and 2% penicillin streptomycin until seeding.

    Incubation:

    Article Title: Calcium-sensing Receptor Modulates Cell Adhesion and Migration via Integrins *
    Article Snippet: .. 96-Well flat bottom plates coated with various concentrations of fibronectin (catalog no. F1141, Sigma) were washed and blocked with 2% filtered BSA solution for 1 h at room temperature. rMTC cells were dissociated, washed, resuspended in assay buffer (serum-free DMEM supplemented with 20 m m HEPES and 0.1% BSA (pH 7.4)), and incubated at 37 °C in a humidified 5% CO2 incubator for 20 min to allow the cells to recover from the process of detachment. .. In parallel, the wells were aspirated, washed, and incubated with 50 μl of 2× DMSO, NPS R-568, or NPS 89636.

    Article Title: Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression
    Article Snippet: .. After, beads were washed three times with MES buffer and incubated over night with fibronectin (Sigma Aldrich F1141) at the concentration of 40 μg ml−1 . ..

    Article Title: Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells
    Article Snippet: .. To facilitate ECM protein coatings, collagen type I (from rat tail, cat.# 1179179, Roche Applied Science, Indianapolis, IN), collagen type IV (from human placenta, cat.# C7521, Sigma Aldrich, St. Louis, MO), and fibronectin (from bovine plasma, cat.# F4759, Sigma Aldrich) were prepared as sterile aqueous solutions (0.1 mg/ml) and independently incubated with silk scaffolds for 1 h at 37°C followed by 1 h of drying in a laminar flow hood at room temperature. .. Uncoated controls were prepared in parallel with incubation in PBS.

    Concentration Assay:

    Article Title: Breast Fibroblasts and ECM Components Modulate Breast Cancer Cell Migration through the Secretion of MMPs in a 3D Microfluidic Co-Culture Model
    Article Snippet: .. For the collagen solution containing fibronectin, fibronectin solution (Sigma-Aldrich, F1141) was added to a final concentration of 100 μg/μL in the collagen. .. For experiments with stromal cells in the matrix, HMFs or CAFs, a final concentration of 500 cells/μL was added to their respective collagen solution.

    Article Title: Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression
    Article Snippet: .. After, beads were washed three times with MES buffer and incubated over night with fibronectin (Sigma Aldrich F1141) at the concentration of 40 μg ml−1 . ..

    Cell Adhesion Assay:

    Article Title: Engineering Attenuated Virulence of a Theileria annulata–Infected Macrophage
    Article Snippet: .. Adhesion assay A 96-well plate was coated with bovine fibronectin (Sigma #F1141), 2 µg/cm2 diluted in double distilled water overnight at 4°C. ..

    other:

    Article Title: Biomechanical Aspects of Actin Bundle Dynamics
    Article Snippet: We detected the time at which the effect of myosin X knock-down was maximal in cells on overall fibronectin-coated glass coverslips.

    Article Title: Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors
    Article Snippet: Briefly, 1,800 μm2 rectangles (aspect ratio 1:5) (RE), 1,800 μm2 circles (BC), and 500 μm2 circles fibronectin (Sigma F1141-2MG) micropatterns (area = 1,800 μm2 and aspect ratio = 1:5) were made on uncoated Ibidi dishes (81151).

    Hood:

    Article Title: Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks
    Article Snippet: .. After activation, the papers were transferred into a sterile 24-well plate under the laminar flow hood and immersed in one of the following surface functionalizing solutions: 10 μg/mL of laminin (L-2020, Sigma-Aldrich) in Neurobasal medium, 20 μg/mL of fibronectin (F1141, Sigma-Aldrich) in Neurobasal medium, or 200 μL of Matrigel (Corning Matrigel Basement Membrane Matrix; 354230, Chemie Brunschwig AG, Switzerland) diluted to 2 mg/mL in ice cold serum-free medium. .. After 45 min of incubation at room temperature, the papers were rinsed with supplemented Neurobasal medium and kept in Neurobasal medium and 2% penicillin streptomycin until seeding.

    Article Title: Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells
    Article Snippet: .. To facilitate ECM protein coatings, collagen type I (from rat tail, cat.# 1179179, Roche Applied Science, Indianapolis, IN), collagen type IV (from human placenta, cat.# C7521, Sigma Aldrich, St. Louis, MO), and fibronectin (from bovine plasma, cat.# F4759, Sigma Aldrich) were prepared as sterile aqueous solutions (0.1 mg/ml) and independently incubated with silk scaffolds for 1 h at 37°C followed by 1 h of drying in a laminar flow hood at room temperature. .. Uncoated controls were prepared in parallel with incubation in PBS.

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  • 93
    Millipore human fibronectin elisa kit
    <t>Fibronectin</t> is present on myeloma-derived exosomes, and a high level of fibronectin correlates with enhanced exosome interaction with target cells. A , result of a single mass spectrometry analysis of selected exosomal proteins from human CAG control (heparanase-low) or aggressive (heparanase-high) myeloma cells. Fibronectin was the protein most enhanced in abundance in exosomes from aggressive cells compared with control. B and C , the amounts of fibronectin associated with exosomes secreted by control or aggressive CAG myeloma cells were quantified by Western blot ( B ) and <t>ELISA</t> ( C ). The results are means ± S.D. of three independent experiments. #, p
    Human Fibronectin Elisa Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fibronectin bovine plasma
    Lateral movement of actin bundles in fibroblasts on homogeneous substrates vs. disk-like patches. (A) Micrograph of a cell membrane-stained (DiI) fibroblast in the late spreading phase on a homogeneous <t>fibronectin</t> coated glass coverslip using TIRF microscopy (scale bar: 15 μm). (B) Time-lapse sequence of the red dashed region of interest in (A) (scale bar: 2 μm). (C) Phase contrast micrograph of a disk-shaped fibroblast (scale bar: 20 μm). Blue circle indicates the fibronectin patch on which the cell adhere. (D) Time-lapse sequence of the red solid region of interest in (C) . An actin bundle, which moves under angle α perpendicular to the cell membrane is highlighted by red arrows (scale bar: 5 μm). Blue arrowhead indicates an actin fold with their width b and distance from the cell membrane d .
    Fibronectin Bovine Plasma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore fibronectin
    EphA4 binds SPAR through a PDZ domain-mediated interaction. A , Freshly prepared hippocampal slices from 3-week-old mice were stimulated with soluble ephrin-A1 Fc (A1 Fc), or Fc as a control, and EphA4 was immunoprecipitated (IP). EphA4 and coimmunoprecipitated SPAR were detected by immunoblotting with the respective antibodies. B , D , Schematic representation of SPAR and EphA4 wild-type and mutant forms. B , GKBD, Guanylate kinase-binding domain. D , Lig BD, Ligand-binding domain; FNIII, <t>fibronectin</t> type III repeat; KD, kinase domain; SAM, sterile-α motif domain; PDZ B, PDZ-domain binding motif; wtEphA4, wild-type EphA4; EphA4K653R, kinase-inactive EphA4; dots indicate tyrosine phosphorylation sites, and the plasma membrane is indicated by a vertical line. C , E , F , The indicated EphA4 constructs and Myc-tagged SPAR constructs were cotransfected in HEK 293T cells. SPAR was immunoprecipitated and detected with anti-Myc antibodies. Coimmunoprecipitated EphA4 was detected by immunoblotting with anti-EphA4 antibodies. Bottom, Similar amounts of EphA4 could be immunoprecipitated from the transfected cells. Lanes between wild-type and mutated EphA4 were digitally removed.
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    Fibronectin is present on myeloma-derived exosomes, and a high level of fibronectin correlates with enhanced exosome interaction with target cells. A , result of a single mass spectrometry analysis of selected exosomal proteins from human CAG control (heparanase-low) or aggressive (heparanase-high) myeloma cells. Fibronectin was the protein most enhanced in abundance in exosomes from aggressive cells compared with control. B and C , the amounts of fibronectin associated with exosomes secreted by control or aggressive CAG myeloma cells were quantified by Western blot ( B ) and ELISA ( C ). The results are means ± S.D. of three independent experiments. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin is present on myeloma-derived exosomes, and a high level of fibronectin correlates with enhanced exosome interaction with target cells. A , result of a single mass spectrometry analysis of selected exosomal proteins from human CAG control (heparanase-low) or aggressive (heparanase-high) myeloma cells. Fibronectin was the protein most enhanced in abundance in exosomes from aggressive cells compared with control. B and C , the amounts of fibronectin associated with exosomes secreted by control or aggressive CAG myeloma cells were quantified by Western blot ( B ) and ELISA ( C ). The results are means ± S.D. of three independent experiments. #, p

    Article Snippet: Fibronectin levels were quantified using a commercially available human fibronectin ELISA kit (Millipore), according to the manufacturer's protocol.

    Techniques: Derivative Assay, Mass Spectrometry, Western Blot, Enzyme-linked Immunosorbent Assay

    Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin on exosomes isolated from multiple myeloma patients facilitates interaction with bone marrow stromal cells. A , characterization of exosomes from serum of treatment naïve multiple myeloma patients. Exosomes were purified from serum of myeloma patients by ExoQuick precipitation followed by isolation using anti-CD63 conjugated beads. Particle size was analyzed by nanoparticle tracking using a NanoSight 300. Histogram shows two lines representing duplicate analyses. B , ELISA quantification of the levels of fibronectin in exosomes isolated from serum of three myeloma patients. C , syndecan-1 and fibronectin are present on the surface of myeloma patient derived exosomes. Exosomes purified from the serum of myeloma patients using ExoQuick precipitation followed by anti-CD63 magnetic bead isolation were subjected to flow cytometry analysis using an affinity-purified polyclonal goat anti-syndecan-1 IgG antibody ( blue ) or mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue ). Normal goat IgG and mouse IgG1 isotype were used as control ( red ), respectively. Note that syndecan-1 (core protein) was detected on the surface of exosomes only after removal of heparan sulfate chains by heparitinase treatment to expose the epitope. D and E , removal of heparan sulfate chains removes most of the fibronectin from exosomes isolated from the serum of myeloma patients. Exosomes isolated from patient serum were either not treated or treated with heparitinase, and fibronectin levels were quantified by ELISA ( D ) and compared by Western blot ( E ). #, p

    Article Snippet: Fibronectin levels were quantified using a commercially available human fibronectin ELISA kit (Millipore), according to the manufacturer's protocol.

    Techniques: Isolation, Purification, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry, Cytometry, Affinity Purification, Western Blot

    Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions *

    doi: 10.1074/jbc.M115.686295

    Figure Lengend Snippet: Fibronectin is present on the exosome surface and its removal inhibits exosome interaction with target cells. A , fibronectin is present on the surface of exosomes. Exosomes from aggressive CAG cells, purified by ultracentrifugation and excluded by an iodixanol cushion, were captured either using anti-CD63 magnetic beads ( left panel ) or using heparin-agarose beads ( right panel ) and subjected to flow cytometry analysis using a mouse monoclonal PE-conjugated anti-fibronectin antibody ( blue histogram ). PE-conjugated isotype-matched antibody was used as a control ( orange histogram ). B , removal of heparan sulfate from the exosome surface removes most of the fibronectin associated with the exosome. Left panel , exosomes were either not treated or treated with bacterial heparitinase (1.5 millunits/ml heparitinase for 3 h at 37 °C followed by addition of fresh enzyme and incubation overnight), a heparan sulfate degrading enzyme, and fibronectin levels were quantified by ELISA. The data are expressed as means ± S.D. #, p

    Article Snippet: Fibronectin levels were quantified using a commercially available human fibronectin ELISA kit (Millipore), according to the manufacturer's protocol.

    Techniques: Purification, Magnetic Beads, Flow Cytometry, Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

    Lateral movement of actin bundles in fibroblasts on homogeneous substrates vs. disk-like patches. (A) Micrograph of a cell membrane-stained (DiI) fibroblast in the late spreading phase on a homogeneous fibronectin coated glass coverslip using TIRF microscopy (scale bar: 15 μm). (B) Time-lapse sequence of the red dashed region of interest in (A) (scale bar: 2 μm). (C) Phase contrast micrograph of a disk-shaped fibroblast (scale bar: 20 μm). Blue circle indicates the fibronectin patch on which the cell adhere. (D) Time-lapse sequence of the red solid region of interest in (C) . An actin bundle, which moves under angle α perpendicular to the cell membrane is highlighted by red arrows (scale bar: 5 μm). Blue arrowhead indicates an actin fold with their width b and distance from the cell membrane d .

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Biomechanical Aspects of Actin Bundle Dynamics

    doi: 10.3389/fcell.2020.00422

    Figure Lengend Snippet: Lateral movement of actin bundles in fibroblasts on homogeneous substrates vs. disk-like patches. (A) Micrograph of a cell membrane-stained (DiI) fibroblast in the late spreading phase on a homogeneous fibronectin coated glass coverslip using TIRF microscopy (scale bar: 15 μm). (B) Time-lapse sequence of the red dashed region of interest in (A) (scale bar: 2 μm). (C) Phase contrast micrograph of a disk-shaped fibroblast (scale bar: 20 μm). Blue circle indicates the fibronectin patch on which the cell adhere. (D) Time-lapse sequence of the red solid region of interest in (C) . An actin bundle, which moves under angle α perpendicular to the cell membrane is highlighted by red arrows (scale bar: 5 μm). Blue arrowhead indicates an actin fold with their width b and distance from the cell membrane d .

    Article Snippet: We detected the time at which the effect of myosin X knock-down was maximal in cells on overall fibronectin-coated glass coverslips.

    Techniques: Staining, Microscopy, Sequencing

    EphA4 binds SPAR through a PDZ domain-mediated interaction. A , Freshly prepared hippocampal slices from 3-week-old mice were stimulated with soluble ephrin-A1 Fc (A1 Fc), or Fc as a control, and EphA4 was immunoprecipitated (IP). EphA4 and coimmunoprecipitated SPAR were detected by immunoblotting with the respective antibodies. B , D , Schematic representation of SPAR and EphA4 wild-type and mutant forms. B , GKBD, Guanylate kinase-binding domain. D , Lig BD, Ligand-binding domain; FNIII, fibronectin type III repeat; KD, kinase domain; SAM, sterile-α motif domain; PDZ B, PDZ-domain binding motif; wtEphA4, wild-type EphA4; EphA4K653R, kinase-inactive EphA4; dots indicate tyrosine phosphorylation sites, and the plasma membrane is indicated by a vertical line. C , E , F , The indicated EphA4 constructs and Myc-tagged SPAR constructs were cotransfected in HEK 293T cells. SPAR was immunoprecipitated and detected with anti-Myc antibodies. Coimmunoprecipitated EphA4 was detected by immunoblotting with anti-EphA4 antibodies. Bottom, Similar amounts of EphA4 could be immunoprecipitated from the transfected cells. Lanes between wild-type and mutated EphA4 were digitally removed.

    Journal: The Journal of Neuroscience

    Article Title: The EphA4 Receptor Regulates Neuronal Morphology through SPAR-Mediated Inactivation of Rap GTPases

    doi: 10.1523/JNEUROSCI.2746-07.2007

    Figure Lengend Snippet: EphA4 binds SPAR through a PDZ domain-mediated interaction. A , Freshly prepared hippocampal slices from 3-week-old mice were stimulated with soluble ephrin-A1 Fc (A1 Fc), or Fc as a control, and EphA4 was immunoprecipitated (IP). EphA4 and coimmunoprecipitated SPAR were detected by immunoblotting with the respective antibodies. B , D , Schematic representation of SPAR and EphA4 wild-type and mutant forms. B , GKBD, Guanylate kinase-binding domain. D , Lig BD, Ligand-binding domain; FNIII, fibronectin type III repeat; KD, kinase domain; SAM, sterile-α motif domain; PDZ B, PDZ-domain binding motif; wtEphA4, wild-type EphA4; EphA4K653R, kinase-inactive EphA4; dots indicate tyrosine phosphorylation sites, and the plasma membrane is indicated by a vertical line. C , E , F , The indicated EphA4 constructs and Myc-tagged SPAR constructs were cotransfected in HEK 293T cells. SPAR was immunoprecipitated and detected with anti-Myc antibodies. Coimmunoprecipitated EphA4 was detected by immunoblotting with anti-EphA4 antibodies. Bottom, Similar amounts of EphA4 could be immunoprecipitated from the transfected cells. Lanes between wild-type and mutated EphA4 were digitally removed.

    Article Snippet: Cells (7 × 104 ) were then allowed to attach to coverslips coated with 10 μg/ml fibronectin (Millipore) or 20 μg/ml poly- l -lysine (Sigma) for 10 min at 37°C in the presence of 10 μg/ml Fc or 10 μg/ml ephrin-A3 Fc.

    Techniques: Mouse Assay, Immunoprecipitation, Mutagenesis, Binding Assay, Ligand Binding Assay, Construct, Transfection