Figure 3.

GREB1-induced hyperactivation of Akt is PI3K dependent. (A) MCF7 cells were transduced with adenovirus expressing GFP or GREB1 and treated with dimethyl sulfoxide (DMSO) or 40 nM GDC0941 simultaneously. Cell lysates were harvested 24 h post-transduction and immunoblot analysis was performed with the indicated antibodies. Immunoblot shown is representative of at least four biological replicates. Histograms depict average phospho-Akt levels normalized to total Akt relative to GFP-overexpressing, DMSO-treated cells ± SD. *P ≤ 0.05 comparison to GFP DMSO; **P ≤ 0.01 comparison to GFP DMSO; ΔΔ P ≤ 0.01 comparison to GREB1 DMSO; ⁺⁺P ≤ 0.01 comparison to GFP GDC0941. (B) MCF7 cells were transduced with lentivirus targeted to a nonspecific control (shNS), PIK3CA, PTEN or PDK1. Following selection, cells were transduced with GFP or GREB1 adenovirus. Cell lysates were harvested 24 h post adenovirus transduction. Following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblot analysis was performed with indicated antibodies. Immunoblot shown is representative of at least four biological replicates. Histograms depict average phospho-Akt levels normalized to total Akt relative to GFP-overexpressing, shNS cells ± SD. *P ≤ 0.05 comparison to shNS GFP, **P ≤ 0.01 comparison to shNS GFP, ⁺P ≤ 0.05 comparison to shNS GREB1, Δ P ≤ 0.05 comparison within same shRNA between GFP and GREB1.