Flt3-ITD inhibits the repressional activity of PLZF. (A) Flt3-ITD up-regulates SRE-LUC reporter. An expression vector for the Flt3-ITD or activated Ha-Ras (800 ng) were cotransfected into 293T cells along with as SRE-LUC reporter (100 ng). The fold induction of transcription was calculated by dividing the luciferase activity, normalized to the internal control renilla plasmid obtained in the presence of the signaling molecules, by the activity obtained from the reporter gene when coexpressed with empty expression vector. The data presented are mean values (± standard deviation) from 4 to 8 independent experiments. (B) PLZF repression is inhibited by the overexpression of Flt3-ITD. The 293T cells were transfected with 100 ng of reporter plasmid; 0, 20, or 200 ng of PLZF expression vector; 800 ng (left) or 1200 ng (right) of Flt3-ITD or Ha-Ras effector plasmids as indicated; and 5 ng of pRL-renilla as an internal control. The fold repression of the reporter gene was calculated from the normalized luciferase activity obtained in the presence of PLZF amount compared with activity obtained in the presence of empty expression vector. The data presented are mean values (± standard deviation) from 4 to 8 independent experiments. (C) Partial restoration of PLZF inhibition by MAPK inhibitor U0126. Experiment performed similar to panel B: 24 hours after transfection, U0126 was added at final concentration at 10 μM in indicated cells and cells were harvested at 48 hours after transfection. The data presented are mean values (± standard deviation) from 4 to 8 independent experiments.