Journal: The Journal of Cell Biology

Article Title: Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

doi:

Figure Lengend Snippet: Cell–cell adhesion via E-cadherin and desmoglein-2 occurs by a cooperative mechanism in both E-variants and TPA-treated R-variants. Monolayers of HCT-8/R1 cells ( R ) were treated with 250 ng/ml TPA as indicated in Materials and Methods. Monolayers of HCT-8/E8 cells ( E ) were not treated with TPA here ( b and d ), although analysis of TPA-treated E-variants yielded essentially identical data (not shown). Both R- and E-variants were analyzed for cell aggregation (increasing particle volume) either after 0 min (broken lines in a and b ; peak positions indicated by 0-min arrows in c and d ) or after 30 min of incubation at 37°C (full lines in all panels). Aggregation for 30 min by R-variants, which were not treated with TPA, yielded curves coinciding with the 0-min curves (not shown). Other curves depicted correspond with the following antibody treatments (see also Materials and Methods). The mAb MB-2 against human E-cadherin (O) was used at an effective concentration (dilution 1:20; a and b ), or at a more ineffective concentration (1:200; c and d , with in c only peak position indicated by an arrow). The mAb 10G11 against desmoglein-2 (□) was also used at an effective concentration (1:2; a and b ), or at an ineffective concentration (1:8; c and d , with in d only peak position indicated by an arrow). These two antibodies, both at the lower concentrations, were also combined as indicated by filled circles (•) in c and d. Upon such combination, a clear-cut synergistic inhibition of cell aggregation was observed for both cell types.

Article Snippet: The following mouse monoclonal antibodies were used: antibodies HECD-1 (Takara Biochemicals, Otsu, Japan) and MB2 ( ) both functionally blocking human E-cadherin, 1G5 against α-catenin ( Becton Dickinson , San Jose, CA), anti–β-catenin against β-catenin (Transduction Laboratories, Lexington, KY), anti-pp120 against p120 cas (Transduction Laboratories), PG 5.1 against plakoglobin and DG 3.10 against desmoglein (both from Cymbus Bioscience Ltd., Southampton, UK), 10G11 against desmoglein-2 (Progen, Heidelberg, Germany), DP 2.15 against desmoplakin I and II ( Boehringer Mannheim , Mannheim, Germany), hVIN-1 against vinculin ( Sigma Immunochemicals, St. Louis, MO), and htr-5 against the human TNF-receptor p55 ( ).

Techniques: Incubation, Concentration Assay, Inhibition

Journal: The Journal of Cell Biology

Article Title: Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

doi:

Figure Lengend Snippet: No major effect on E-cadherin, but formation of typical tight junctions and spot desmosomes upon TPA treatment of α-catenin–negative variants of HCT-8. Cells were double stained for E-cadherin ( a–c ) and occludin ( d–f ), or stained for desmoglein-2 ( g–i ). Each panel comprises an extended depth view by overlaying several confocal X-Y sections ( top ) and confocal X-Z sections of the same field ( bottom ). Cells analyzed were: epithelioid HCT-8/E8 cells ( a , d , g ), untreated HCT-8/R1 cells ( b , e , h ), and TPA-treated (250 ng/ml for 3 h) HCT-8/R1 cells ( c , f , i ). Bar, 14 μm.

Article Snippet: The following mouse monoclonal antibodies were used: antibodies HECD-1 (Takara Biochemicals, Otsu, Japan) and MB2 ( ) both functionally blocking human E-cadherin, 1G5 against α-catenin ( Becton Dickinson , San Jose, CA), anti–β-catenin against β-catenin (Transduction Laboratories, Lexington, KY), anti-pp120 against p120 cas (Transduction Laboratories), PG 5.1 against plakoglobin and DG 3.10 against desmoglein (both from Cymbus Bioscience Ltd., Southampton, UK), 10G11 against desmoglein-2 (Progen, Heidelberg, Germany), DP 2.15 against desmoplakin I and II ( Boehringer Mannheim , Mannheim, Germany), hVIN-1 against vinculin ( Sigma Immunochemicals, St. Louis, MO), and htr-5 against the human TNF-receptor p55 ( ).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

doi:

Figure Lengend Snippet: Relocalization of desmosomal proteins to cell–cell contacts upon TPA treatment of α-catenin–negative variants of HCT-8. Cells were stained for plakoglobin ( a , b , and c ), desmoglein-2 ( d , e , and f ), or desmoplakin ( g , h , and i ). In the untreated epithelioid variant HCT-8/E8, desmosomal proteins were localized at the cell–cell contacts ( a , d , and g ). HCT-8/R1 cultures were either untreated ( b , e , and h ) or treated for 3 h with 250 ng/ml TPA ( c , f , and i ). Similar observations were done for other R-variants of HCT-8. Bar, 10 μm.

Article Snippet: The following mouse monoclonal antibodies were used: antibodies HECD-1 (Takara Biochemicals, Otsu, Japan) and MB2 ( ) both functionally blocking human E-cadherin, 1G5 against α-catenin ( Becton Dickinson , San Jose, CA), anti–β-catenin against β-catenin (Transduction Laboratories, Lexington, KY), anti-pp120 against p120 cas (Transduction Laboratories), PG 5.1 against plakoglobin and DG 3.10 against desmoglein (both from Cymbus Bioscience Ltd., Southampton, UK), 10G11 against desmoglein-2 (Progen, Heidelberg, Germany), DP 2.15 against desmoplakin I and II ( Boehringer Mannheim , Mannheim, Germany), hVIN-1 against vinculin ( Sigma Immunochemicals, St. Louis, MO), and htr-5 against the human TNF-receptor p55 ( ).

Techniques: Staining, Variant Assay

Journal: The Journal of Cell Biology

Article Title: Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

doi:

Figure Lengend Snippet: TPA induces exposition of desmoglein-2 molecules at the cell surface in α-catenin–negative variants of HCT-8. Monolayers of the epithelioid HCT-8/E8 ( E ) and of the round cell variant HCT-8/R1 ( R ) were treated for the indicated times with 250 ng/ml TPA, and then labeled with NHS-biotin for 30 min and extracted with lysis buffer. Biotinylated proteins were purified by adsorbing to avidin-agarose beads and analyzed by Western blotting using antibodies to E-cadherin (HECD-1) or desmoglein (DG 3.10).

Article Snippet: The following mouse monoclonal antibodies were used: antibodies HECD-1 (Takara Biochemicals, Otsu, Japan) and MB2 ( ) both functionally blocking human E-cadherin, 1G5 against α-catenin ( Becton Dickinson , San Jose, CA), anti–β-catenin against β-catenin (Transduction Laboratories, Lexington, KY), anti-pp120 against p120 cas (Transduction Laboratories), PG 5.1 against plakoglobin and DG 3.10 against desmoglein (both from Cymbus Bioscience Ltd., Southampton, UK), 10G11 against desmoglein-2 (Progen, Heidelberg, Germany), DP 2.15 against desmoplakin I and II ( Boehringer Mannheim , Mannheim, Germany), hVIN-1 against vinculin ( Sigma Immunochemicals, St. Louis, MO), and htr-5 against the human TNF-receptor p55 ( ).

Techniques: Variant Assay, Labeling, Lysis, Purification, Avidin-Biotin Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

doi:

Figure Lengend Snippet: TPA does not modify the detergent solubility of E-cadherin, β-catenin, plakoglobin, and desmoglein-2. Monolayers of HCT-8/E8 ( E ) and HCT-8/R1 ( R ) were either untreated or treated with 250 ng/ml TPA for 3 h as indicated. Detergent (2.5% NP-40) soluble and insoluble fractions were prepared and analyzed by Western blotting using antibodies to the antigens indicated at the right.

Article Snippet: The following mouse monoclonal antibodies were used: antibodies HECD-1 (Takara Biochemicals, Otsu, Japan) and MB2 ( ) both functionally blocking human E-cadherin, 1G5 against α-catenin ( Becton Dickinson , San Jose, CA), anti–β-catenin against β-catenin (Transduction Laboratories, Lexington, KY), anti-pp120 against p120 cas (Transduction Laboratories), PG 5.1 against plakoglobin and DG 3.10 against desmoglein (both from Cymbus Bioscience Ltd., Southampton, UK), 10G11 against desmoglein-2 (Progen, Heidelberg, Germany), DP 2.15 against desmoplakin I and II ( Boehringer Mannheim , Mannheim, Germany), hVIN-1 against vinculin ( Sigma Immunochemicals, St. Louis, MO), and htr-5 against the human TNF-receptor p55 ( ).

Techniques: Solubility, Western Blot

Journal: The Journal of Cell Biology

Article Title: Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

doi:

Figure Lengend Snippet: Transfection of αN-catenin cDNA into the round cell variant HCT-8/E11R1 induces desmosome formation. Transfected cell clone HRpCαN2 was fixed and double stained with anti-αN-catenin mAb ( a ) and anti-desmoglein mAb ( b ). Note the different location of the two antigens, with the immunoreactivity of desmoglein being punctate as might be expected for typical desmosomes. Bar, 5 μm.

Article Snippet: The following mouse monoclonal antibodies were used: antibodies HECD-1 (Takara Biochemicals, Otsu, Japan) and MB2 ( ) both functionally blocking human E-cadherin, 1G5 against α-catenin ( Becton Dickinson , San Jose, CA), anti–β-catenin against β-catenin (Transduction Laboratories, Lexington, KY), anti-pp120 against p120 cas (Transduction Laboratories), PG 5.1 against plakoglobin and DG 3.10 against desmoglein (both from Cymbus Bioscience Ltd., Southampton, UK), 10G11 against desmoglein-2 (Progen, Heidelberg, Germany), DP 2.15 against desmoplakin I and II ( Boehringer Mannheim , Mannheim, Germany), hVIN-1 against vinculin ( Sigma Immunochemicals, St. Louis, MO), and htr-5 against the human TNF-receptor p55 ( ).

Techniques: Transfection, Variant Assay, Staining