Journal:

Article Title: The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERK-Mediated Actin Spike Formation: Deregulation in Cancer Cells 1

doi:

Figure Lengend Snippet: Cell-matrix adhesion assembly on fibronectin is enhanced during the progression of colon cancer. (A) Visualization of AA/C1/SB10 carcinoma cell-matrix adhesion assembly 1 hour after plating on fibronectin. Cells were fixed and stained for vinculin. The solid arrow represents a spread cell with prominent vinculin-containing protrusions, while the broken arrow represents an adherent, but non-spread cell which is tethered to the surface by a ring of small vinculin-containing structures. Scale bar, 25 µm. (B) Cell-matrix adhesion assembly on fibronectin in AA/C1 and AA/C1/SB10 cells was quantitated by counting the percentage of cells with prominent adhesions in >500 adherent cells for the indicated times. Values are mean±SD from triplicate wells. (C) Cell-matrix adhesion assembly on collagen was measured by counting the percentage of cells with prominent focal adhesions in >500 cells. Values are mean±SD from triplicate wells. (D) Cell attachment to fibronectin and collagen was measured after 60 minutes. Values are mean±SD from quadruplicate wells.

Article Snippet: Permanox chamber slides were coated with fibronectin (10 μ g/ml; Becton Dickinson, Oxford, UK) by overnight incubation at 4°C.

Techniques: Staining, Cell Attachment Assay

Journal:

Article Title: The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERK-Mediated Actin Spike Formation: Deregulation in Cancer Cells 1

doi:

Figure Lengend Snippet: Cell-matrix adhesion assembly on fibronectin is integrin-mediated. (A) Cell-matrix adhesion assembly in AA/C1/SB10 cells on fibronectin was measured by counting the percentage of cells with prominent adhesions in >500 cells in the presence of inhibitory integrin antibodies as indicated. Values are mean±SD from triplicate wells. (B) Cell-matrix adhesion assembly on αvβ6 antibody and anti-mouse IgG coated chambers (top panel) or anti-mouse IgG alone-coated chambers (bottom panel). (C) Cell surface integrin expression was determined in the AA/C1 and AA/C1/SB10 cells following FACS analysis using integrin antibodies as described in Materials and Methods section. Relative fluorescence values were determined using the arbitrary geometric mean values calculated for the individual histograms, from which the non-specific fluorescence was deducted. Non-specific fluorescence was measured in the absence of antibody. Values are taken from a representative experiment in a series of three.

Article Snippet: Permanox chamber slides were coated with fibronectin (10 μ g/ml; Becton Dickinson, Oxford, UK) by overnight incubation at 4°C.

Techniques: Expressing, Fluorescence

Journal:

Article Title: The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERK-Mediated Actin Spike Formation: Deregulation in Cancer Cells 1

doi:

Figure Lengend Snippet: FAK phosphorylation is required for integrin-mediated adhesion assembly. (A) FAK phosphorylation in cells either held in suspension (Sus), plated on fibronectin (FN), or poly-l-lysine (PL) for 1 hour. FAK was immunoprecipitated from cell lysates and tyrosine-phosphorylated FAK visualized by blotting and probing with PY20, a general phosphotyrosine antibody (left hand, top panel). The same filters were reprobed with anti-FAK antibody (left hand, bottom panel). FAK phosphorylated on tyrosine-397 was immunoprecipitated from AA/C1/SB10 cell lysates using a phospho-specific antibody and immunoprecipitated proteins visualized by probing with an anti-FAK antibody (right panel). (B) AA/C1/SB10 cells were transfected with Myc-tagged wtFAK or FAK-Y397F and trypsinized after 24 hours before plating on fibronectin. After 1 hour, the cells were fixed and stained with an antibody to vinculin and the Myc epitope tag. Cell-matrix adhesion assembly was quantitated in the non-transfected and transfected cells. Values are mean±SD from triplicate wells with >500 transfected cells. (C) Visualization of Myc-tagged proteins following double staining with anti-Myc and anti-vinculin antibodies. Scale bars, 25 µm.

Article Snippet: Permanox chamber slides were coated with fibronectin (10 μ g/ml; Becton Dickinson, Oxford, UK) by overnight incubation at 4°C.

Techniques: Phospho-proteomics, Suspension, Immunoprecipitation, Transfection, Staining, Double Staining

Journal:

Article Title: The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERK-Mediated Actin Spike Formation: Deregulation in Cancer Cells 1

doi:

Figure Lengend Snippet: Localization of activated ERK in cell-matrix adhesions. (A) Cells were fixed after 1 hour on fibronectin and co-stained with anti-phospho-ERK antibodies and an anti-vinculin antibody. Scale bars, 25 µm. (B) AA/C1/SB10 cells were transfected with HA-tagged ERK and trypsinized after 24 hours before plating on fibronectin. After 1 hour, the cells were fixed and stained with an antibody to HA. Scale bars, 25 µm. (C) AA/C1/SB10 cells treated with PD098059 were fixed after 1 hour on fibronectin and co-stained with anti-phospho-ERK antibodies and an anti-vinculin antibody. (D) AA/C1/SB10 cells were transfected with Myc-tagged FAK (Y397F) and, after 24 hours, trypsinized before plating on fibronectin. After 1 hour, the cells were fixed and stained with an antibody to phospho-ERK and the Myc epitope tag. Control cells were untransfected cells in the same culture. Scale bars, 25 µm.

Article Snippet: Permanox chamber slides were coated with fibronectin (10 μ g/ml; Becton Dickinson, Oxford, UK) by overnight incubation at 4°C.

Techniques: Staining, Transfection, Control

Journal:

Article Title: The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERK-Mediated Actin Spike Formation: Deregulation in Cancer Cells 1

doi:

Figure Lengend Snippet: ERK is required for integrin-mediated adhesion assembly. (A) Cell-matrix adhesion assembly on fibronectin in the presence of PD098059 (25 µM) or UO126 (20 µM) was measured by counting the percentage of cells with prominent adhesions in >500 adherent cells. Values are mean±SD from triplicate wells. (B) Visualization of cell-matrix adhesions in untreated and inhibitor-treated cells stained with anti-vinculin antibody. Scale bars, 25 µm. Quantitation of cells with protruding cell-matrix adhesions from >500 cells (lower panel). Values are mean±SD from triplicate wells.

Article Snippet: Permanox chamber slides were coated with fibronectin (10 μ g/ml; Becton Dickinson, Oxford, UK) by overnight incubation at 4°C.

Techniques: Staining, Quantitation Assay

Journal:

Article Title: The Protrusive Phase and Full Development of Integrin-Dependent Adhesions in Colon Epithelial Cells Require FAK- and ERK-Mediated Actin Spike Formation: Deregulation in Cancer Cells 1

doi:

Figure Lengend Snippet: Actin rearrangements during cell-matrix adhesion assembly. (A) Cells were fixed after 1 hour on fibronectin and co-stained with phalloidin-FITC and either antibodies to talin or vinculin. (B) As above, except AA/C1/SB10 cells were pre-treated with cytochalasin D (0.25 µg/ ml) or UO126 (20 µM) for 15 minutes prior to plating. Scale bars, 25 µm.

Article Snippet: Permanox chamber slides were coated with fibronectin (10 μ g/ml; Becton Dickinson, Oxford, UK) by overnight incubation at 4°C.

Techniques: Staining