fibronectin  (Roche)


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    Structured Review

    Roche fibronectin
    Visusalizing cell-induced FN fibrillogenesis by AFM and fluorescence microscopy. (A) Individual MEF cells were seeded on a homogenous FN layer adsorbed onto mica disks for 1 h and then imaged by continuous AFM contact mode scanning. Representative image frames (error channel) extracted from the timelapse series (see Supplementary Movie 1 ) show the gradual creation of FN nanofibrils at sites of membrane retraction (arrows). An insert in the left panel shows a magnified view (height image) of the created FN nanofibrils of the area denoted by the dashed box. Size of the AFM timelapse images 10 × 10 μm 2 , full range of the height scale (insert) is 15 nm. (B) Mouse embryonic fibroblasts (MEFs) expressing incubated on Alexa Fluor 488-labeled <t>fibronectin</t> (FN-AF488) for 1 h, fixed and immunostained for paxillin. Merged (left panel) and higher magnification single and merged channel images (center and right panels) corresponding to the area denoted by the dashed box. (C) Complementary AFM height and TIRF microscopy images of MEFs incubated on FN-AF488 for 1h after fixation and staining for F-actin. The areas denoted by the dashed boxes (left panels) shown at high magnification (right panels).
    Fibronectin, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fibronectin/product/Roche
    Average 93 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    fibronectin - by Bioz Stars, 2020-09
    93/100 stars

    Images

    1) Product Images from "Controlling Fibronectin Fibrillogenesis Using Visible Light"

    Article Title: Controlling Fibronectin Fibrillogenesis Using Visible Light

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2020.00149

    Visusalizing cell-induced FN fibrillogenesis by AFM and fluorescence microscopy. (A) Individual MEF cells were seeded on a homogenous FN layer adsorbed onto mica disks for 1 h and then imaged by continuous AFM contact mode scanning. Representative image frames (error channel) extracted from the timelapse series (see Supplementary Movie 1 ) show the gradual creation of FN nanofibrils at sites of membrane retraction (arrows). An insert in the left panel shows a magnified view (height image) of the created FN nanofibrils of the area denoted by the dashed box. Size of the AFM timelapse images 10 × 10 μm 2 , full range of the height scale (insert) is 15 nm. (B) Mouse embryonic fibroblasts (MEFs) expressing incubated on Alexa Fluor 488-labeled fibronectin (FN-AF488) for 1 h, fixed and immunostained for paxillin. Merged (left panel) and higher magnification single and merged channel images (center and right panels) corresponding to the area denoted by the dashed box. (C) Complementary AFM height and TIRF microscopy images of MEFs incubated on FN-AF488 for 1h after fixation and staining for F-actin. The areas denoted by the dashed boxes (left panels) shown at high magnification (right panels).
    Figure Legend Snippet: Visusalizing cell-induced FN fibrillogenesis by AFM and fluorescence microscopy. (A) Individual MEF cells were seeded on a homogenous FN layer adsorbed onto mica disks for 1 h and then imaged by continuous AFM contact mode scanning. Representative image frames (error channel) extracted from the timelapse series (see Supplementary Movie 1 ) show the gradual creation of FN nanofibrils at sites of membrane retraction (arrows). An insert in the left panel shows a magnified view (height image) of the created FN nanofibrils of the area denoted by the dashed box. Size of the AFM timelapse images 10 × 10 μm 2 , full range of the height scale (insert) is 15 nm. (B) Mouse embryonic fibroblasts (MEFs) expressing incubated on Alexa Fluor 488-labeled fibronectin (FN-AF488) for 1 h, fixed and immunostained for paxillin. Merged (left panel) and higher magnification single and merged channel images (center and right panels) corresponding to the area denoted by the dashed box. (C) Complementary AFM height and TIRF microscopy images of MEFs incubated on FN-AF488 for 1h after fixation and staining for F-actin. The areas denoted by the dashed boxes (left panels) shown at high magnification (right panels).

    Techniques Used: Fluorescence, Microscopy, Expressing, Incubation, Labeling, Staining

    2) Product Images from "Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model"

    Article Title: Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21113898

    Quantitative analysis of the protein expression using the scoring method on the immunohistochemistry stained images. ( a ) Fibronectin, ( b ) filaggrin, ( c ) involucrin, and ( d ) keratin.
    Figure Legend Snippet: Quantitative analysis of the protein expression using the scoring method on the immunohistochemistry stained images. ( a ) Fibronectin, ( b ) filaggrin, ( c ) involucrin, and ( d ) keratin.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    Immunohistochemistry stained images for fibronectin of 3D cell cultured sample for 3 days air exposure using culture media-treated CLLE concentrations of ( a ) 0 μg/mL, ( b ) 50 μg/mL, and ( c ) 250 μg/mL; for 5 days air exposure using culture media-treated CLLE concentrations of ( d ) 0 μg/mL, ( e ) 50 μg/mL, and ( f ) 250 μg/mL; and for 7 days air exposure using culture media-treated CLLE concentrations of ( g ) 0 μg/mL, ( h ) 50 μg/mL, and ( i ) 250 μg/mL.
    Figure Legend Snippet: Immunohistochemistry stained images for fibronectin of 3D cell cultured sample for 3 days air exposure using culture media-treated CLLE concentrations of ( a ) 0 μg/mL, ( b ) 50 μg/mL, and ( c ) 250 μg/mL; for 5 days air exposure using culture media-treated CLLE concentrations of ( d ) 0 μg/mL, ( e ) 50 μg/mL, and ( f ) 250 μg/mL; and for 7 days air exposure using culture media-treated CLLE concentrations of ( g ) 0 μg/mL, ( h ) 50 μg/mL, and ( i ) 250 μg/mL.

    Techniques Used: Immunohistochemistry, Staining, Cell Culture

    3) Product Images from "All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules"

    Article Title: All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules

    Journal: Cell Adhesion & Migration

    doi: 10.4161/cam.4.3.11682

    (A) MDA-MB-231 cells (300,000 cells/1.5 ml) grown in presence (lane 2) and in absence (lane 1) of 20 µM ATRA for 48 hrs. Both control and ATRA treated cells were then allowed to grow in presence of 20 µg/ml fibronectin for 2 h in SFCM.
    Figure Legend Snippet: (A) MDA-MB-231 cells (300,000 cells/1.5 ml) grown in presence (lane 2) and in absence (lane 1) of 20 µM ATRA for 48 hrs. Both control and ATRA treated cells were then allowed to grow in presence of 20 µg/ml fibronectin for 2 h in SFCM.

    Techniques Used: Multiple Displacement Amplification

    (A) MDA-MB-231 cells grown in presence (Treated) and in absence (Control) of 20 µM ATRA were allowed to bind with different concentration of fibronectin (25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.125 µg/ml, 1.56 µg/ml)
    Figure Legend Snippet: (A) MDA-MB-231 cells grown in presence (Treated) and in absence (Control) of 20 µM ATRA were allowed to bind with different concentration of fibronectin (25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.125 µg/ml, 1.56 µg/ml)

    Techniques Used: Multiple Displacement Amplification, Concentration Assay

    4) Product Images from "Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells"

    Article Title: Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0234638

    Characterization of naïve human HSCs grown on SiOn-covered and uncovered 3D PDMS and collagen- or fibronectin-covered and uncovered PS. Human HSCs were cultured for 14 DIV in HSC medium supplemented with 1 mM VPA on uncovered 2D PS, 3D PDMS, collagen- or fibronectin-covered 2D PS as well as SiOn-covered 3D PDMS. CD34+ and CD34+/ CD38-/ CD45RA-/ CD49f+/ CD90+ cells were determined by flow cytometric analyses using a combination of specific antibodies and vital cells were selected using DAPI. CFUs were counted after additional 14 days of incubation in multi-lineage CFU medium. The amplification of all cells and CD34+ cells, the percentage and absolute number of CD34+ cells are depicted for three different donors (A, C) as their mean ± SD. The amplification, percentage and the absolute cell number of CD34+/ CD38-/ CD45RA-/ CD49f+/ CD90+ cells are depicted for three different donors (B, D) as their mean ± SD. Total numbers of CFU per 1x10 4 seeded human HSCs are depicted for three different donors (E) as their mean ± SD. Colony-forming unit-granulocyte/erythroid/megakaryocyte/monocyte (CFU-GEMM), colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E), colony-forming unit-macrophage (CFU-M), colony-forming unit-granulocyte (CFU-G). *p
    Figure Legend Snippet: Characterization of naïve human HSCs grown on SiOn-covered and uncovered 3D PDMS and collagen- or fibronectin-covered and uncovered PS. Human HSCs were cultured for 14 DIV in HSC medium supplemented with 1 mM VPA on uncovered 2D PS, 3D PDMS, collagen- or fibronectin-covered 2D PS as well as SiOn-covered 3D PDMS. CD34+ and CD34+/ CD38-/ CD45RA-/ CD49f+/ CD90+ cells were determined by flow cytometric analyses using a combination of specific antibodies and vital cells were selected using DAPI. CFUs were counted after additional 14 days of incubation in multi-lineage CFU medium. The amplification of all cells and CD34+ cells, the percentage and absolute number of CD34+ cells are depicted for three different donors (A, C) as their mean ± SD. The amplification, percentage and the absolute cell number of CD34+/ CD38-/ CD45RA-/ CD49f+/ CD90+ cells are depicted for three different donors (B, D) as their mean ± SD. Total numbers of CFU per 1x10 4 seeded human HSCs are depicted for three different donors (E) as their mean ± SD. Colony-forming unit-granulocyte/erythroid/megakaryocyte/monocyte (CFU-GEMM), colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E), colony-forming unit-macrophage (CFU-M), colony-forming unit-granulocyte (CFU-G). *p

    Techniques Used: Cell Culture, Incubation, Amplification

    5) Product Images from "Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity"

    Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity

    Journal: Pancreas

    doi: 10.1097/MPA.0000000000000270

    Prdx1 associates with phospho-p38 MAPK in cell protrusions. A, Immunoprecipitation of Prdx1 from S2-013 cells cultured on fibronectin. Immunoprecipitates were examined on Western blots probed with antibodies against phospho-ASK1, phospho-p38 MAPK, and phospho-JNK. Rabbit immunoglobulin G was used as an isotype control. B, Immunocytochemical staining in S2-013 cells cultured on fibronectin; anti-Prdx1 (green) and anti–phospho-p38 MAPK (red) antibodies were used to label endogenous proteins. Arrows, Prdx1 colocalized with phospho-p38 MAPK in cell protrusions. Blue, DAPI staining (bar, 10 μm). C, Western blotting of total cell lysates probing for phospho-ASK1, phospho-p38 MAPK, and phospho-JNK in scrambled control and Prdx1 RNAi S2-013 cells cultured on fibronectin.
    Figure Legend Snippet: Prdx1 associates with phospho-p38 MAPK in cell protrusions. A, Immunoprecipitation of Prdx1 from S2-013 cells cultured on fibronectin. Immunoprecipitates were examined on Western blots probed with antibodies against phospho-ASK1, phospho-p38 MAPK, and phospho-JNK. Rabbit immunoglobulin G was used as an isotype control. B, Immunocytochemical staining in S2-013 cells cultured on fibronectin; anti-Prdx1 (green) and anti–phospho-p38 MAPK (red) antibodies were used to label endogenous proteins. Arrows, Prdx1 colocalized with phospho-p38 MAPK in cell protrusions. Blue, DAPI staining (bar, 10 μm). C, Western blotting of total cell lysates probing for phospho-ASK1, phospho-p38 MAPK, and phospho-JNK in scrambled control and Prdx1 RNAi S2-013 cells cultured on fibronectin.

    Techniques Used: Immunoprecipitation, Cell Culture, Western Blot, Staining

    Prdx1 localizes in cell protrusions. A, Confocal Z stack shows the accumulation of Prdx1 (green) in membrane protrusions and nuclear DAPI staining (blue) in S2-013 cells. Arrows, Prdx1 localized in cell protrusions. The lower and light panels in the confocal Z stack show a vertical cross section (yellow lines) through the cells (bar, 10 μm). B, S2-013 cells were incubated on fibronectin and immunocytochemically stained using anti-Prdx1 antibody (green) and phalloidin (red). Actin filaments were labeled by phalloidin. Arrows, Prdx1 localized in cell protrusions. Blue, DAPI staining (bar, 10 μm). C, A confluent S2-013 cell monolayer was wounded. After 4 hours, anti-Prdx1 antibody (green) and phalloidin (red) were used to immunostain the cells. Arrows, Prdx1 at the leading edges of migrating cells. Blue, DAPI staining (bar, 10 μm). D, Immunohistochemical staining with anti-Prdx1 antibody in S2-013 primary pancreatic tumors in mice. Arrows, Prdx1 localized at the cell membranes (representative sections, original magnification ×200).
    Figure Legend Snippet: Prdx1 localizes in cell protrusions. A, Confocal Z stack shows the accumulation of Prdx1 (green) in membrane protrusions and nuclear DAPI staining (blue) in S2-013 cells. Arrows, Prdx1 localized in cell protrusions. The lower and light panels in the confocal Z stack show a vertical cross section (yellow lines) through the cells (bar, 10 μm). B, S2-013 cells were incubated on fibronectin and immunocytochemically stained using anti-Prdx1 antibody (green) and phalloidin (red). Actin filaments were labeled by phalloidin. Arrows, Prdx1 localized in cell protrusions. Blue, DAPI staining (bar, 10 μm). C, A confluent S2-013 cell monolayer was wounded. After 4 hours, anti-Prdx1 antibody (green) and phalloidin (red) were used to immunostain the cells. Arrows, Prdx1 at the leading edges of migrating cells. Blue, DAPI staining (bar, 10 μm). D, Immunohistochemical staining with anti-Prdx1 antibody in S2-013 primary pancreatic tumors in mice. Arrows, Prdx1 localized at the cell membranes (representative sections, original magnification ×200).

    Techniques Used: Staining, Incubation, Labeling, Immunohistochemistry, Mouse Assay

    Prdx1 plays a role in increasing the activity of p38 MAPK. A, S2-013 cells were treated with SB203580, and then the cells were incubated on fibronectin. Western blotting of steady-state levels of the cells was performed using antibodies against phospho-ASK1, phospho-p38 MAPK, phospho-MAPKAP2, and phospho-JNK. B, Scrambled control S2-013 cells or Prdx1 -knockdown S2-013 cells treated with SB203580 were plated on Matrigel invasion chambers. Invading cells in 4 fields per group were counted. Data were derived from 3 independent experiments. Columns, Mean; bars, SD. * P
    Figure Legend Snippet: Prdx1 plays a role in increasing the activity of p38 MAPK. A, S2-013 cells were treated with SB203580, and then the cells were incubated on fibronectin. Western blotting of steady-state levels of the cells was performed using antibodies against phospho-ASK1, phospho-p38 MAPK, phospho-MAPKAP2, and phospho-JNK. B, Scrambled control S2-013 cells or Prdx1 -knockdown S2-013 cells treated with SB203580 were plated on Matrigel invasion chambers. Invading cells in 4 fields per group were counted. Data were derived from 3 independent experiments. Columns, Mean; bars, SD. * P

    Techniques Used: Activity Assay, Incubation, Western Blot, Derivative Assay

    Prdx1 and phospho-p38 MAPK accumulated in cell protrusions induce the further formation of membrane protrusions. A, Scrambled control S2-013 cells or Prdx1 -knockdown S2-013 cells were incubated on fibronectin and immunocytochemically stained with anti-Prdx1 antibody (green), phalloidin (red), and antibody against anti–phospho-p38 MAPK (upper panels) or anti–phospho-MAPKAP2 (lower panels) (violet). Actin filaments were labeled by phalloidin. Arrows, Prdx1 colocalized with phospho-p38 MAPK in cell protrusions of control cells. Yellow arrows, Phospho-MAPKAP2 accumulated in cell protrusions. White arrowheads, Fibronectin-mediated peripheral actin structures in control cells. Yellow arrowheads, Decreased peripheral actin structures by suppression of Prdx1. Blue, DAPI staining (bars, 10 μm). B, Quantification of data shown in A; the values represent the number of scrambled control S2-013 cells or Prdx1 -knockdown S2-013 cells with cell protrusions in which peripheral actin structures were increased. All cells in 4 fields per group were scored. Data were derived from 3 independent experiments. Columns, Mean; bars, SD. * P
    Figure Legend Snippet: Prdx1 and phospho-p38 MAPK accumulated in cell protrusions induce the further formation of membrane protrusions. A, Scrambled control S2-013 cells or Prdx1 -knockdown S2-013 cells were incubated on fibronectin and immunocytochemically stained with anti-Prdx1 antibody (green), phalloidin (red), and antibody against anti–phospho-p38 MAPK (upper panels) or anti–phospho-MAPKAP2 (lower panels) (violet). Actin filaments were labeled by phalloidin. Arrows, Prdx1 colocalized with phospho-p38 MAPK in cell protrusions of control cells. Yellow arrows, Phospho-MAPKAP2 accumulated in cell protrusions. White arrowheads, Fibronectin-mediated peripheral actin structures in control cells. Yellow arrowheads, Decreased peripheral actin structures by suppression of Prdx1. Blue, DAPI staining (bars, 10 μm). B, Quantification of data shown in A; the values represent the number of scrambled control S2-013 cells or Prdx1 -knockdown S2-013 cells with cell protrusions in which peripheral actin structures were increased. All cells in 4 fields per group were scored. Data were derived from 3 independent experiments. Columns, Mean; bars, SD. * P

    Techniques Used: Incubation, Staining, Labeling, Derivative Assay

    6) Product Images from "Nonmuscle Myosin IIA-Dependent Force Inhibits Cell Spreading and Drives F-Actin Flow"

    Article Title: Nonmuscle Myosin IIA-Dependent Force Inhibits Cell Spreading and Drives F-Actin Flow

    Journal:

    doi: 10.1529/biophysj.106.084806

    Retrograde F-actin flow is driven by NMM-IIA, not NMM-IIB. ( A ) A differential interference contrast image taken from a time-lapse movie of fibronectin-coated 2.7- μ m magnetic beads transported on the surface of a MEF cell. White arrow depicts the
    Figure Legend Snippet: Retrograde F-actin flow is driven by NMM-IIA, not NMM-IIB. ( A ) A differential interference contrast image taken from a time-lapse movie of fibronectin-coated 2.7- μ m magnetic beads transported on the surface of a MEF cell. White arrow depicts the

    Techniques Used: Flow Cytometry, Magnetic Beads

    NMM-IIA is essential for focal adhesion and stress fiber formation. Control cells and NMM-IIA-knockdown cells were spread on coverslips coated with 10 μ g/ml fibronectin for 90 min at 37°C, and then fixed and subjected to immunostaining.
    Figure Legend Snippet: NMM-IIA is essential for focal adhesion and stress fiber formation. Control cells and NMM-IIA-knockdown cells were spread on coverslips coated with 10 μ g/ml fibronectin for 90 min at 37°C, and then fixed and subjected to immunostaining.

    Techniques Used: Immunostaining

    Quantitative comparisons of the focal adhesions in control and NMM-IIA-knockdown cells. On fibronectin substrate, the area of focal adhesions in NMM-IIA-deficient cells is significantly smaller than controls. The focal adhesion area in RPTP-C6 cells (82.0
    Figure Legend Snippet: Quantitative comparisons of the focal adhesions in control and NMM-IIA-knockdown cells. On fibronectin substrate, the area of focal adhesions in NMM-IIA-deficient cells is significantly smaller than controls. The focal adhesion area in RPTP-C6 cells (82.0

    Techniques Used:

    NMM-IIA regulates cell spreading of mouse embryonic fibroblasts. ( A ) Sequential TIRF images of a Calcein AM-labeled MEF cell spreading on fibronectin substrate. Panels B , C , and D are plots of cell area over a course of time. NMM-IIB +/+
    Figure Legend Snippet: NMM-IIA regulates cell spreading of mouse embryonic fibroblasts. ( A ) Sequential TIRF images of a Calcein AM-labeled MEF cell spreading on fibronectin substrate. Panels B , C , and D are plots of cell area over a course of time. NMM-IIB +/+

    Techniques Used: Labeling

    Related Articles

    Immunohistochemistry:

    Article Title: Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model
    Article Snippet: .. For immunohistochemistry (IHC) staining, the slides were incubated with primary antibodies specific to Fibronectin (ab2413), Involucrin (ab53112), Cytokeratin (ab76318), Laminin 5 (ab14509), and Filaggrin (ab81468), separately overnight at 4 °C and processed using Benchmark XT Auto-stainer System (Roche), following the manufacturer’s instructions. ..

    Protease Inhibitor:

    Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
    Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

    Labeling:

    Article Title: Controlling Fibronectin Fibrillogenesis Using Visible Light
    Article Snippet: .. Fibronectin Labeling and Surface Coating Lyophilized human plasma FN (Roche, Grenzach-Wyhlen, Germany) was resuspended in sterile water and stored at −80°C. .. Fibronectin was labeled by conjugating Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 633 succinimidyl (NHS) ester dyes to primary amines of FN using the Alexa Fluor Protein Labeling Kit .

    Immunoprecipitation:

    Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
    Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

    Incubation:

    Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
    Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

    Article Title: Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model
    Article Snippet: .. For immunohistochemistry (IHC) staining, the slides were incubated with primary antibodies specific to Fibronectin (ab2413), Involucrin (ab53112), Cytokeratin (ab76318), Laminin 5 (ab14509), and Filaggrin (ab81468), separately overnight at 4 °C and processed using Benchmark XT Auto-stainer System (Roche), following the manufacturer’s instructions. ..

    other:

    Article Title: Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells
    Article Snippet: Cells were seeded into blank wells (2D PS) for control, SiOn-covered or uncovered 2D and 3D PDMS structures, as well as fibronectin (R & D Biosystem, 5 μg/cm2 ) or collagen (Roche, 5 μg/cm2) covered PS.

    Article Title: All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules
    Article Snippet: Fibronectin (440 kD) and protein G agarose was purchased from Roche, Germany.

    Lysis:

    Article Title: Peroxiredoxin 1 Promotes Pancreatic Cancer Cell Invasion by Modulating p38 MAPK Activity
    Article Snippet: .. Immunoprecipitation S2-013 cells were incubated on fibronectin for 5 hours and lysed in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl2 , 0.5% NP-40, protease inhibitor cocktail tablets [Roche], and phosphatase inhibitor cocktail [Nacalai, Kyoto, Japan]). .. Lysates were immunoprecipitated with Dynabeads Protein G (Dynal, Oslo, Norway) and with anti-Prdx1 antibody or with normal rabbit immunoglobulin G for 2 hours at 4°C.

    Staining:

    Article Title: Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model
    Article Snippet: .. For immunohistochemistry (IHC) staining, the slides were incubated with primary antibodies specific to Fibronectin (ab2413), Involucrin (ab53112), Cytokeratin (ab76318), Laminin 5 (ab14509), and Filaggrin (ab81468), separately overnight at 4 °C and processed using Benchmark XT Auto-stainer System (Roche), following the manufacturer’s instructions. ..

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  • 93
    Roche fibronectin
    Visusalizing cell-induced FN fibrillogenesis by AFM and fluorescence microscopy. (A) Individual MEF cells were seeded on a homogenous FN layer adsorbed onto mica disks for 1 h and then imaged by continuous AFM contact mode scanning. Representative image frames (error channel) extracted from the timelapse series (see Supplementary Movie 1 ) show the gradual creation of FN nanofibrils at sites of membrane retraction (arrows). An insert in the left panel shows a magnified view (height image) of the created FN nanofibrils of the area denoted by the dashed box. Size of the AFM timelapse images 10 × 10 μm 2 , full range of the height scale (insert) is 15 nm. (B) Mouse embryonic fibroblasts (MEFs) expressing incubated on Alexa Fluor 488-labeled <t>fibronectin</t> (FN-AF488) for 1 h, fixed and immunostained for paxillin. Merged (left panel) and higher magnification single and merged channel images (center and right panels) corresponding to the area denoted by the dashed box. (C) Complementary AFM height and TIRF microscopy images of MEFs incubated on FN-AF488 for 1h after fixation and staining for F-actin. The areas denoted by the dashed boxes (left panels) shown at high magnification (right panels).
    Fibronectin, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fibronectin/product/Roche
    Average 93 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    fibronectin - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    84
    Roche human fibronectin coated surface
    PH-domain-mediated membrane recruitment and diffusion of kindlin-2 is crucial during cell spreading. ( a ) Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4h on <t>fibronectin.</t> Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). ( b ) Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). GFP-paxillin: 141 cells; GFP-paxillin + mEos2-kindlin-2-WT: 312 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 311 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 309 cells; GFP-paxillin + mEos2-kindlin-2-QW615/615AA: 284 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 289 cells. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). ( c ) TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of cell area ( d ) and total area of FAs ( e ) of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-ΔPH (orange) or mEos2-kindlin-2-ΔPH-CAAX (green) and 4h after seeding on fibronectin. Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 93 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 134 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 120 cells; The results correspond to pooled data from four independent experiments. Between 23 and 42 cells per experiment were used for the kindlin-2-ΔPH or kindlin-2-ΔPH-CAAX conditions (kindlin-2-WT: between 17 and 26 cells per experiment). Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01
    Human Fibronectin Coated Surface, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche fibronectin coated coverslips
    DEPDC1B Silencing Perturbs Adhesion and Actin Cytoskeleton Dynamics in G2 Phase (A and B) FA dynamics were observed using GFP-paxillin (A) or vinculin (B) as reporters in control (Ctrl) or DEPDC1B-silenced (1B-KD1) HeLa cells synchronized in early G2 phase (D-THY plus 4 hr release). (A) The mean number, size (area, μm 2 ), and the duration of FAs, determined by total internal reflection fluorescence (TIRF) microscopy, in control (Ctrl, blue) and DEPDC1B-silenced cells (KD-1B, red) are shown. (B) The average number and area (μm 2 ) of FAs per cell, determined by confocal microscopy using vinculin staining, are shown. (C) The actin cytoskeleton (FITC-phalloidin) and the activation of myosin light chain (phospho-MLC2-Ser19) were analyzed by immunofluorescence and confocal microscopy in HeLa cells synchronized in G2 phase. The percentage (mean ± SEM of three experiments) of cells with high/low phospho-MLC2 staining is reported. (D) Western blot analysis shows levels of phospho-MLC2, phospho-Cofilin (Ser3) upon DEPDC1B-KD. Vinculin, total MLC2, and Cofilin were used as loading controls. In parallel, RhoA activity was measured by GST-RBD pull-down (right panel). As positive control, the lysate was activated by GTPγS stimulation. An asterisk marks a nonspecific band detected by the anti-RhoA antibody. The densitometry analysis of active RhoA in two experiments is shown. (E) IMR90 and MCF10A cells were analyzed in G2 phase for actin cytoskeleton and FA dynamics as described in (B) and (C). Western blot analysis shows levels of phospho-MLC2 (Ser19) upon DEPDC1B silencing. (F) Cell spreading dynamics, on <t>fibronectin</t> substrate, of G2 synchronized HeLa cells was monitored by Real Time Cell Analyzer ( Atienzar et al., 2011 ). Graph shows the normalized cell index, as a measure of cell-covered area after replating. Asterisks in graphs mark significant values (p
    Fibronectin Coated Coverslips, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Visusalizing cell-induced FN fibrillogenesis by AFM and fluorescence microscopy. (A) Individual MEF cells were seeded on a homogenous FN layer adsorbed onto mica disks for 1 h and then imaged by continuous AFM contact mode scanning. Representative image frames (error channel) extracted from the timelapse series (see Supplementary Movie 1 ) show the gradual creation of FN nanofibrils at sites of membrane retraction (arrows). An insert in the left panel shows a magnified view (height image) of the created FN nanofibrils of the area denoted by the dashed box. Size of the AFM timelapse images 10 × 10 μm 2 , full range of the height scale (insert) is 15 nm. (B) Mouse embryonic fibroblasts (MEFs) expressing incubated on Alexa Fluor 488-labeled fibronectin (FN-AF488) for 1 h, fixed and immunostained for paxillin. Merged (left panel) and higher magnification single and merged channel images (center and right panels) corresponding to the area denoted by the dashed box. (C) Complementary AFM height and TIRF microscopy images of MEFs incubated on FN-AF488 for 1h after fixation and staining for F-actin. The areas denoted by the dashed boxes (left panels) shown at high magnification (right panels).

    Journal: Frontiers in Molecular Biosciences

    Article Title: Controlling Fibronectin Fibrillogenesis Using Visible Light

    doi: 10.3389/fmolb.2020.00149

    Figure Lengend Snippet: Visusalizing cell-induced FN fibrillogenesis by AFM and fluorescence microscopy. (A) Individual MEF cells were seeded on a homogenous FN layer adsorbed onto mica disks for 1 h and then imaged by continuous AFM contact mode scanning. Representative image frames (error channel) extracted from the timelapse series (see Supplementary Movie 1 ) show the gradual creation of FN nanofibrils at sites of membrane retraction (arrows). An insert in the left panel shows a magnified view (height image) of the created FN nanofibrils of the area denoted by the dashed box. Size of the AFM timelapse images 10 × 10 μm 2 , full range of the height scale (insert) is 15 nm. (B) Mouse embryonic fibroblasts (MEFs) expressing incubated on Alexa Fluor 488-labeled fibronectin (FN-AF488) for 1 h, fixed and immunostained for paxillin. Merged (left panel) and higher magnification single and merged channel images (center and right panels) corresponding to the area denoted by the dashed box. (C) Complementary AFM height and TIRF microscopy images of MEFs incubated on FN-AF488 for 1h after fixation and staining for F-actin. The areas denoted by the dashed boxes (left panels) shown at high magnification (right panels).

    Article Snippet: Fibronectin Labeling and Surface Coating Lyophilized human plasma FN (Roche, Grenzach-Wyhlen, Germany) was resuspended in sterile water and stored at −80°C.

    Techniques: Fluorescence, Microscopy, Expressing, Incubation, Labeling, Staining

    Glial cell initial adhesion and proliferation in mixed cultures in DMEMs medium on fibronectin coating. A - Phase contrast imaging of glial cells at the border of stiff (42 kPa) and soft regions (1.1 kPa) at 21 DIV of a “concentric” pattern of rigidity. Scale bar: 50 µm. B - Evolution of the cell density over time (DIV) on stiff (black squares) and soft (white circles) regions. **** denotes that the two means are significantly different, p (stiff vs. soft at 3 and at 10 DIV)

    Journal: bioRxiv

    Article Title: Glial cell mechanosensitivity is reversed by adhesion cues

    doi: 10.1101/865303

    Figure Lengend Snippet: Glial cell initial adhesion and proliferation in mixed cultures in DMEMs medium on fibronectin coating. A - Phase contrast imaging of glial cells at the border of stiff (42 kPa) and soft regions (1.1 kPa) at 21 DIV of a “concentric” pattern of rigidity. Scale bar: 50 µm. B - Evolution of the cell density over time (DIV) on stiff (black squares) and soft (white circles) regions. **** denotes that the two means are significantly different, p (stiff vs. soft at 3 and at 10 DIV)

    Article Snippet: We employed fibronectin (FN, from human plasma, Roche Applied Science, Switzerland, ref 11080938001) or poly-L-lysine and laminin (PLL, P2636; LN, L2020, Sigma-Aldrich, St. Louis, USA).

    Techniques: Imaging

    Glial cells from pure cultures in the presence of gradients of rigidity in DMEMs medium on fibronectin coating. A - Glial cells grown on “concentric” rigidity pattern align perpendicular to the gradient of rigidity at the border between the stiff and soft regions (17 DIV). B - Glial cells are deformed by subcellular rigidity patterns (stiff stripes of 5 µm, 40 kPa, alternating with soft stripes of 20 µm, 12 kPa, 2 DIV). Scale bars: 50 µm.

    Journal: bioRxiv

    Article Title: Glial cell mechanosensitivity is reversed by adhesion cues

    doi: 10.1101/865303

    Figure Lengend Snippet: Glial cells from pure cultures in the presence of gradients of rigidity in DMEMs medium on fibronectin coating. A - Glial cells grown on “concentric” rigidity pattern align perpendicular to the gradient of rigidity at the border between the stiff and soft regions (17 DIV). B - Glial cells are deformed by subcellular rigidity patterns (stiff stripes of 5 µm, 40 kPa, alternating with soft stripes of 20 µm, 12 kPa, 2 DIV). Scale bars: 50 µm.

    Article Snippet: We employed fibronectin (FN, from human plasma, Roche Applied Science, Switzerland, ref 11080938001) or poly-L-lysine and laminin (PLL, P2636; LN, L2020, Sigma-Aldrich, St. Louis, USA).

    Techniques:

    Rigidity sensitivity in DMEMs of glial cells from mixed cultures is enhanced compared to pure cultures. A - Comparison of the normalized initial adhesion of glial cells from pure cultures on the different coatings (“FN”: fibronectin, “PLL/LN”: poly-L-lysine/laminin) and rigidities (“Soft”: 1.1 kPa, “Stiff”: 42 kPa). B - Normalized initial proliferation rates show that glial cells proliferate faster in mixed culture conditions. C - Comparison of the evolution of the cell density in pure and mixed cultures on the stiff regions coated with FN.

    Journal: bioRxiv

    Article Title: Glial cell mechanosensitivity is reversed by adhesion cues

    doi: 10.1101/865303

    Figure Lengend Snippet: Rigidity sensitivity in DMEMs of glial cells from mixed cultures is enhanced compared to pure cultures. A - Comparison of the normalized initial adhesion of glial cells from pure cultures on the different coatings (“FN”: fibronectin, “PLL/LN”: poly-L-lysine/laminin) and rigidities (“Soft”: 1.1 kPa, “Stiff”: 42 kPa). B - Normalized initial proliferation rates show that glial cells proliferate faster in mixed culture conditions. C - Comparison of the evolution of the cell density in pure and mixed cultures on the stiff regions coated with FN.

    Article Snippet: We employed fibronectin (FN, from human plasma, Roche Applied Science, Switzerland, ref 11080938001) or poly-L-lysine and laminin (PLL, P2636; LN, L2020, Sigma-Aldrich, St. Louis, USA).

    Techniques:

    Glial cell initial adhesion and proliferation in pure cultures on fibronectin coated substrates. A - Immunofluo-rescence images of a glial cell culture in DMEMs medium: the frontier (solid white line) delineates the soft, inner region and the stiff, outside area. The dashed white lines mark out the immunofluorescence images in the rigidity pattern. Red: phalloidin, actin. Green: N-Cadherin, cell-cell adhesions. Blue: Hoechst, nuclei. Cells are fixed at 12 DIV. Scale bar: 300 µm. B - Evolution of the cell density in DMEMs medium over time (days in vitro, DIV) on stiff (black squares) and soft (white circles) regions. ** denotes that the two means are significantly different, p (stiff vs. soft) = 0.0050 (2 DIV); *, p = 0.0187 (stiff, 2 DIV vs. between 6 and 17 DIV); ****, p

    Journal: bioRxiv

    Article Title: Glial cell mechanosensitivity is reversed by adhesion cues

    doi: 10.1101/865303

    Figure Lengend Snippet: Glial cell initial adhesion and proliferation in pure cultures on fibronectin coated substrates. A - Immunofluo-rescence images of a glial cell culture in DMEMs medium: the frontier (solid white line) delineates the soft, inner region and the stiff, outside area. The dashed white lines mark out the immunofluorescence images in the rigidity pattern. Red: phalloidin, actin. Green: N-Cadherin, cell-cell adhesions. Blue: Hoechst, nuclei. Cells are fixed at 12 DIV. Scale bar: 300 µm. B - Evolution of the cell density in DMEMs medium over time (days in vitro, DIV) on stiff (black squares) and soft (white circles) regions. ** denotes that the two means are significantly different, p (stiff vs. soft) = 0.0050 (2 DIV); *, p = 0.0187 (stiff, 2 DIV vs. between 6 and 17 DIV); ****, p

    Article Snippet: We employed fibronectin (FN, from human plasma, Roche Applied Science, Switzerland, ref 11080938001) or poly-L-lysine and laminin (PLL, P2636; LN, L2020, Sigma-Aldrich, St. Louis, USA).

    Techniques: Cell Culture, Immunofluorescence, In Vitro

    PH-domain-mediated membrane recruitment and diffusion of kindlin-2 is crucial during cell spreading. ( a ) Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4h on fibronectin. Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). ( b ) Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). GFP-paxillin: 141 cells; GFP-paxillin + mEos2-kindlin-2-WT: 312 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 311 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 309 cells; GFP-paxillin + mEos2-kindlin-2-QW615/615AA: 284 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 289 cells. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). ( c ) TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of cell area ( d ) and total area of FAs ( e ) of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-ΔPH (orange) or mEos2-kindlin-2-ΔPH-CAAX (green) and 4h after seeding on fibronectin. Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 93 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 134 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 120 cells; The results correspond to pooled data from four independent experiments. Between 23 and 42 cells per experiment were used for the kindlin-2-ΔPH or kindlin-2-ΔPH-CAAX conditions (kindlin-2-WT: between 17 and 26 cells per experiment). Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01

    Journal: bioRxiv

    Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

    doi: 10.1101/2020.08.06.239731

    Figure Lengend Snippet: PH-domain-mediated membrane recruitment and diffusion of kindlin-2 is crucial during cell spreading. ( a ) Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4h on fibronectin. Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). ( b ) Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). GFP-paxillin: 141 cells; GFP-paxillin + mEos2-kindlin-2-WT: 312 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 311 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 309 cells; GFP-paxillin + mEos2-kindlin-2-QW615/615AA: 284 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 289 cells. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). ( c ) TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of cell area ( d ) and total area of FAs ( e ) of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-ΔPH (orange) or mEos2-kindlin-2-ΔPH-CAAX (green) and 4h after seeding on fibronectin. Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 93 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 134 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 120 cells; The results correspond to pooled data from four independent experiments. Between 23 and 42 cells per experiment were used for the kindlin-2-ΔPH or kindlin-2-ΔPH-CAAX conditions (kindlin-2-WT: between 17 and 26 cells per experiment). Where indicated, statistical significance was obtained using two-tailed, non-parametric Mann–Whitney rank sum test. The resulting P values are indicated as follows: ns: P > 0.05; *: 0.01

    Article Snippet: Cells were then seeded on human fibronectin-coated surface (fibronectin: 10 μg/ml, Roche).

    Techniques: Diffusion-based Assay, Knock-Out, Transfection, Expressing, Negative Control, Two Tailed Test, MANN-WHITNEY

    DEPDC1B Silencing Perturbs Adhesion and Actin Cytoskeleton Dynamics in G2 Phase (A and B) FA dynamics were observed using GFP-paxillin (A) or vinculin (B) as reporters in control (Ctrl) or DEPDC1B-silenced (1B-KD1) HeLa cells synchronized in early G2 phase (D-THY plus 4 hr release). (A) The mean number, size (area, μm 2 ), and the duration of FAs, determined by total internal reflection fluorescence (TIRF) microscopy, in control (Ctrl, blue) and DEPDC1B-silenced cells (KD-1B, red) are shown. (B) The average number and area (μm 2 ) of FAs per cell, determined by confocal microscopy using vinculin staining, are shown. (C) The actin cytoskeleton (FITC-phalloidin) and the activation of myosin light chain (phospho-MLC2-Ser19) were analyzed by immunofluorescence and confocal microscopy in HeLa cells synchronized in G2 phase. The percentage (mean ± SEM of three experiments) of cells with high/low phospho-MLC2 staining is reported. (D) Western blot analysis shows levels of phospho-MLC2, phospho-Cofilin (Ser3) upon DEPDC1B-KD. Vinculin, total MLC2, and Cofilin were used as loading controls. In parallel, RhoA activity was measured by GST-RBD pull-down (right panel). As positive control, the lysate was activated by GTPγS stimulation. An asterisk marks a nonspecific band detected by the anti-RhoA antibody. The densitometry analysis of active RhoA in two experiments is shown. (E) IMR90 and MCF10A cells were analyzed in G2 phase for actin cytoskeleton and FA dynamics as described in (B) and (C). Western blot analysis shows levels of phospho-MLC2 (Ser19) upon DEPDC1B silencing. (F) Cell spreading dynamics, on fibronectin substrate, of G2 synchronized HeLa cells was monitored by Real Time Cell Analyzer ( Atienzar et al., 2011 ). Graph shows the normalized cell index, as a measure of cell-covered area after replating. Asterisks in graphs mark significant values (p

    Journal: Developmental Cell

    Article Title: DEPDC1B Coordinates De-adhesion Events and Cell-Cycle Progression at Mitosis

    doi: 10.1016/j.devcel.2014.09.009

    Figure Lengend Snippet: DEPDC1B Silencing Perturbs Adhesion and Actin Cytoskeleton Dynamics in G2 Phase (A and B) FA dynamics were observed using GFP-paxillin (A) or vinculin (B) as reporters in control (Ctrl) or DEPDC1B-silenced (1B-KD1) HeLa cells synchronized in early G2 phase (D-THY plus 4 hr release). (A) The mean number, size (area, μm 2 ), and the duration of FAs, determined by total internal reflection fluorescence (TIRF) microscopy, in control (Ctrl, blue) and DEPDC1B-silenced cells (KD-1B, red) are shown. (B) The average number and area (μm 2 ) of FAs per cell, determined by confocal microscopy using vinculin staining, are shown. (C) The actin cytoskeleton (FITC-phalloidin) and the activation of myosin light chain (phospho-MLC2-Ser19) were analyzed by immunofluorescence and confocal microscopy in HeLa cells synchronized in G2 phase. The percentage (mean ± SEM of three experiments) of cells with high/low phospho-MLC2 staining is reported. (D) Western blot analysis shows levels of phospho-MLC2, phospho-Cofilin (Ser3) upon DEPDC1B-KD. Vinculin, total MLC2, and Cofilin were used as loading controls. In parallel, RhoA activity was measured by GST-RBD pull-down (right panel). As positive control, the lysate was activated by GTPγS stimulation. An asterisk marks a nonspecific band detected by the anti-RhoA antibody. The densitometry analysis of active RhoA in two experiments is shown. (E) IMR90 and MCF10A cells were analyzed in G2 phase for actin cytoskeleton and FA dynamics as described in (B) and (C). Western blot analysis shows levels of phospho-MLC2 (Ser19) upon DEPDC1B silencing. (F) Cell spreading dynamics, on fibronectin substrate, of G2 synchronized HeLa cells was monitored by Real Time Cell Analyzer ( Atienzar et al., 2011 ). Graph shows the normalized cell index, as a measure of cell-covered area after replating. Asterisks in graphs mark significant values (p

    Article Snippet: For adhesion experiments, cells were plated on fibronectin-coated coverslips (final concentration: 10 μg/ml) and monitored by immunofluorescence (FITC-phalloidin) or by Real-Time Cell Analyzer Technology (xCELLigence Roche).

    Techniques: Fluorescence, Microscopy, Confocal Microscopy, Staining, Activation Assay, Immunofluorescence, Western Blot, Activity Assay, Positive Control