Figure 2
The PI3K/Akt pathway does not participate in the dexamethasone-mediated protection from TNF-α-dependent cell death in MCF-7 cells: (A) Cell cultures were treated with TNF-α (10 ng ml-1), dexamethasone (Dex) (10 μM) or both for 20 min: Western blotts of whole-cell extracts were performed with anti-phosphorylated Akt (pAkt) specific antibodies (upper panel): After stripping, membranes were re-blotted against total Akt (tAkt) (lower panel): (B) Parental cells and cells from ΔP85-expressing MCF-7 clones (A6, A8 and A10) were treated with TNF-α (10 ng ml-1) for 20 min and total (tAkt) and phosphorylated (pAkt) Akt were determined as in A: IκB protein degradation (C) and NF-κB nuclear translocation (D) were determined by Western blot and EMSA respectively in parental and ΔP85 expressing cell clone A6 treated with TNF-α (10 ng ml-1) for 20 min. Each blot is representative of three independent experiments. Below the blots in A, B, C, and D the bar graphs indicate the relative density of each lane with respect to control, which has an arbitrary value of 1: (E) Dexamethasone protection against TNF-α-mediated cytotoxicity was evaluated in parental MCF-7 cells (MCF-7) and three independent clones expressing the ΔP85 protein (A6, A8 and A10): Cell viability of clones incubated either with TNF-α (10 ng ml-1) alone or with TNF-α and Dex (10 μM) for 48 hrs was determined. Values are mean ± SD from three independent experiments performed in triplicate. * indicates p < 0.01 vs TNF.