FOXO3a and BIM expression and function. (A) Reduction of FoxO3a activity. SUDHL4, SUDHL10, and OCI-LY3 cells were treated with indicated concentration of AZD6244 for 24 hours. Cell lysates were subjected to Western blotting using p-FoxO3a and FoxO3a antibodies. (B) Induction of proapoptotic proteins by AZD6244. SUDHL4 and OCI-LY-19 cells were treated with 200nM AZD6244 for indicated periods of time. PUMA and BIM proteins were detected by immunoblotting using specific antibodies. (C) Increase in proapoptotic proteins PUMA and BIM in PBMCs from DLBCL patients by Western blotting after AZD6244 treatment for 18 hours. Actin was used as a loading control for all blots. (D) OCI-LY3 and (E) SUDHL4 cells were transfected with BIM siRNA or scrambled siRNA, using Amaxa nucleofection kit, followed by incubation with 100nM or 200nM AZD6244 for 48 hours. Knockdown of BIM is shown by Western blot. Apoptosis was measured by annexin V/PI staining followed by flow cytometry. (F) OCI-LY3 cells were transduced with BIM shRNA using lentivirus system. Stably transduced cells that were subsequently transfected with Bcl-2 or Mcl-1 expressing plasmids. After 24-hour transfection, cells were treated with 200nM of AZD6244 for 48 hours and annexin V/PI staining and analyzed by flow cytometry.