Figure 3

Inhibition of NF-κB activity abrogates the dexamethasone protection from TNF-α mediated cell death: (A) MCF-7 cells were treated as in figure 2A and DNA NF-κB binding was evaluated in nuclear extracts by EMSA: (B) Wild type MCF-7 cells and MCF-7 clones 1 and 8 expressing a dominant negative IκB protein (dnIκB) or the empty vector (GFP) were treated with TNF-α (10 ng ml^-1) for 20 min: After cell lysis, endogenous (wtIκB) and recombinant (dnIκB) proteins were detected in whole cell extract by Western blot: Total Akt (tAkt) is shown as loading control: (C) Indicated cell cultures were treated as in 2A: Then, NF-κB nuclear translocation was evaluated by EMSA. Each blot is representative of three independent experiments. Below the blots in A, B, and C the bar graphs indicate the relative density of each lane with respect to control, which has an arbitrary value of 1. In C the first bar graph represents the relative densities for dnIκBα, while the second bar graph represents the relative densities for wtIκBα: (D) The indicated cell clones were incubated with TNF-α (10 ng ml^-1) alone or TNF-α (10 ng ml^-1) and Dex (10 μM) for 48 hrs and cell viability was determined. Values are mean ± SD from three independent experiments performed in triplicate. * indicates p < 0.01 with respect to MCF-7 survival after TNF-α treatment.