Journal: The Journal of Cell Biology

Article Title: E-Cadherin–dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27 KIP1

doi:

Figure Lengend Snippet: E-cadherin inhibits growth factor–mediated downregulation of p27 through a phosphatase-dependent mechanism. ( a ) Mitogens are required for growth stimulation in the presence of E-cadherin–neutralizing antibodies. Nontreated HT29 cells ( control ) were grown in serum-free medium for 48 h. Treated cells were grown in the presence of 2 μg/ml SHE78-7 antibody (+ SHE ), 10% FCS (+ FCS ), or 50 ng/ml TGF-α (+ TGF - α ), either alone or in various combinations. To prevent TGF-α activity, cultures were treated with c225-neutralizing anti-EGRF antibody. All factors were added to three-dimensional cell cultures at the time of cell plating. After pulsing with [ 3 H]thymidine, radioactivity incorporated into DNA was measured and expressed as fold increase in counts over nontreated control cells. The corresponding levels of p27 as determined by immunoblotting are shown in the inset. ( b ) Inhibition of phosphatase activity lowers p27 levels in aggregated HT29 cells. HT29 cells plated in three-dimensional culture in the presence of either FCS or TGF-α were treated with 50 μM sodium orthovanadate (+ Van ). 48 h later, cells were collected and levels of p27 were determined by immunoblotting.

Article Snippet: All cells were cultured in their normal growth medium containing 10% FCS, with the following exceptions: in the experiment that examined mitogen-dependence of growth stimulation by E-cadherin antibodies, cells were plated in serum-free medium or medium containing either 10% FCS or 50 ng/ml TGF-α (R&D Systems, Inc., Minneapolis, MN).

Techniques: Activity Assay, Radioactivity, Western Blot, Inhibition

Journal: The Journal of Cell Biology

Article Title: E-Cadherin–dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27 KIP1

doi:

Figure Lengend Snippet: Inhibiting PI3K activity or generating reactive oxygen species elevates p27 protein levels and reduces [ 3 H]thymidine uptake of E-cadherin antibody-dispersed HT29 cells. Cells plated into three-dimensional cuture in the presence of TGF-α and SHE78-7 antibody were treated with either ( a ) the PI3K inhibitor LY294002 ( LY ) or ( b ) the antioxidant N -acetyl- l -cysteine (NAC). [ 3 H]thymidine incorporation was measured after 24 h. The inset for both a and b shows the corresponding levels of p27 protein as determined by immunoblotting.

Article Snippet: All cells were cultured in their normal growth medium containing 10% FCS, with the following exceptions: in the experiment that examined mitogen-dependence of growth stimulation by E-cadherin antibodies, cells were plated in serum-free medium or medium containing either 10% FCS or 50 ng/ml TGF-α (R&D Systems, Inc., Minneapolis, MN).

Techniques: Activity Assay, Western Blot