Journal: Cell Death Discovery

Article Title: MUC1 O -glycosylation contributes to anoikis resistance in epithelial cancer cells

doi: 10.1038/cddiscovery.2017.44

Figure Lengend Snippet: Effect of shRNA C1GT suppression on antibody accessibility to cell surface anoikis-initiating molecules. The shRNA C1GT transfected or shRNA control cells from the MUC1- positive F3 cells were incubated with antibodies against E-cadherin, integrin β 1, CD44 or Fas. After washing and application of FITC-conjugated secondary antibody, the cells were analysed by flow cytometry ( a , solid and dotted lines show the peak positions of control and shRNA C1GT transfected cells, respectively. Dotted line shifted to the left or right of the solid line reveals reduction or increase of molecule expression). Mean florescence intensity of the antibody binding are shown in b . Cells were also lysed and analysed by immunoblotting ( c ) for the expression of these molecules with the same antibodies as in a . ** P <0.01, *** P <0.001.

Article Snippet: Antibodies against CD44 (BBA10), integrin β 1 (MAB17782), E-cadherin (MAB1838), Fas (AF2267) and Fas-L (AF126) were from R&D Systems (Abingdon, UK).

Techniques: shRNA, Transfection, Control, Incubation, Flow Cytometry, Expressing, Binding Assay, Western Blot

Journal: Cell Death Discovery

Article Title: MUC1 O -glycosylation contributes to anoikis resistance in epithelial cancer cells

doi: 10.1038/cddiscovery.2017.44

Figure Lengend Snippet: Effect of shRNA C1GT suppression increases antibody accessibility to cell surface anoikis-initiating molecules and enhances Fas-L-induced caspase-8 activity in SW620 cells. shRNA C1GT transfected or shRNA control transfected SW620 cells were incubated with antibodies against E-cadherin, integrin β 1, CD44 or Fas. After application of FITC-conjugated secondary antibody, the cells were analysed by flow cytometry ( a , solid and dotted lines show the peak positions of control and shRNA C1GT transfected cells, respectively. Dotted line shifted to the left or right of the solid line reveals reduction or increase of molecule expression). Mean (±S.D.) florescence intensity of the antibody bindings are shown in b . Cells were also lysed and analysed for the expression of these molecules by immunoblotting with the same antibodies ( c ). In d , the shRNA control and shRNA C1GT transfected SW620 cells were cultured in the presence or absence of 100 ng/ml recombinant Fas-L in suspension and the cell caspase-8 activity was determined after 2 h by Caspase-8-Glo kit. Data are expressed as mean±S.E.M. of triplicate determination of three experiments, *** P <0.001.

Article Snippet: Antibodies against CD44 (BBA10), integrin β 1 (MAB17782), E-cadherin (MAB1838), Fas (AF2267) and Fas-L (AF126) were from R&D Systems (Abingdon, UK).

Techniques: shRNA, Activity Assay, Transfection, Control, Incubation, Flow Cytometry, Expressing, Western Blot, Cell Culture, Recombinant, Suspension