Effect of AKT signaling. (A) Western blotting showing p-AKT and AKT expression. SUDHL4 and OCI-LY3 cells were treated with indicated concentrations of AZD6244 for 24 hours. Whole cell lysates were used to determine protein expression by Western blotting using specific antibodies against p-AKT and AKT. (B) AKT knockdown using AKT siRNA. OCI-LY3 cell were transfected with AKT siRNA or scrambled siRNA using Amaxa nucleofection kit. Knockdown of AKT is shown by Western blotting. After 24 hour transfection, cells were treated with AZD6244 for 48 hours. Apoptosis was measured by annexin V/PI by flow cytometry. (C) Chemical blockade of the PI3/AKT pathway. OCI-LY3 cells were pretreated with 20μM of LY294002 (LY) for 1 hour followed by incubation with 200nM AZD6244 for 48 hours. Apoptosis was measured by annexin V/PI staining followed by flow cytometry. (D) Constitutive activation of AKT. OCI-LY3 cells were transfected with either constitutively active AKT (Myr-Akt) or vector (pcDNA) alone. After selection in neomycin for 14 days, positively selected cells were treated with indicated concentration of AZD6244 for 48 hours. Apoptosis was measured by annexin V/PI staining followed by flow cytometry.