Inhibition of Akt activation via α₄β₁ enhances cell death in G1E-ER2.

(A) Reduced activation of Akt at Ser473 via α₄β₁ (H296) adhesion. Factor-starved G1E-ER2 cells were left unstimulated or cultured on FN peptides mediating adhesion via α₄β₁ (H296) or α₅β₁ (CH271) in the presence of SCF for various time points. Subsequently, at various times, cell lysates were collected and subjected to Western blot analysis with a rabbit antiphospho-Akt antibody that specifically detects phosphorylated S473. Bottom panel shows total Akt in each lane. The position of the activated Akt (pAkt) is indicated. Upper panel (bars) quantitatively demonstrates the relative phosphorylation of Akt. Data are presented relative to the phosphorylation of Akt after 120 minutes (taken as 100). Bars denote the mean relative phosphorylation (± SEM) of at least 3 independent experiments. Asterisk indicatesP < .05 α₄β₁ (H296) versus α₅β₁ (CH271). (B) G1E-ER2 cells were cultured on BSA or FN peptide CH271 or H296 in the presence of SCF and the PI-3K/Akt inhibitor (wortmannin) for 48 hours. Cell death was quantitated by performing annexin and PI staining as described in “Materials and methods.” Bars denote the percentage of total cell death (± SD) of 2 independent experiments performed in replicates of 3. Asterisk indicates P < .05 BSA, α₅β₁ (CH271), α₄β₁ (H296) (wortmannin) versus BSA, α₅β₁ (CH271), α₄β₁ (H296) (no inhibitor). (C,D) G1E-ER2 cells were cultured on FN peptides in the presence of SCF and analyzed at the indicated time points. Cell lysates were collected and subjected to Western blot analysis with an anti–Bcl-2 antibody. The position of Bcl-2 and Bcl-x_L is indicated.