Effect of AKT inhibitor AKTVIII on ligand-independent proliferation, colony formation, and erythroid differentiation mediated by KIT ECD mutants. (A) Effect of AKTVIII on proliferation of KIT variants expressing Ba/F3 cells. Ba/F3 cells expressing WT or KIT mutants were plated in 96-well plates and grown for 48 hours with or without mSCF or mIL-3 and in the presence of 2.5μM AKTVIII. Cell growth was assessed by measuring mitochondrial conversion of WST-1 into blue formazan dye with a spectrophotometer. Values are presented relative to cell proliferation in the absence of AKTVIII. Data are mean ± SD of triplicates. Results shown are representative of 3 independent experiments. (B) Effect of AKTVIII on activation of AKT and its downstream substrates in Ba/F3-Del^417-419insY cells. Cells were serum starved for 5 hours and treated with 0 to 2.5μM AKTVIII for 1 hour before lysis. Phosphorylation of AKT, GSK3β, mTOR, STAT3, and KIT was analyzed by Western blotting using the indicated activation-specific antibodies. The blot was stripped and reprobed with different antibodies as loading controls. (C) Inhibition of Epo-induced BFU-E formation mediated by Del^417-419insY mutant. EML cells expressing WT-KIT, Del^417-419insY mutant, or MyrAKT were plated in Epo and/or SCF containing methylcellulose in the presence or absence of 0.06μM AKTVIII. Results are percentage of BFU-E or CFU-GM colonies in the presence of AKTVIII relative to those in the absence of AKTVIII. Data are mean ± SD of duplicates. Experiments were performed twice.