Figure 5

CITED2 knockdown mediates decrease in AKT-signaling in a p53/PHLDA3/BCL2-dependent manner (a) p53 co-IP of sorted control- or shCITED2-transduced MOLM-13 cells with subsequent western blot analysis for MDM2 (left panel). Right panel indicates quantified MDM2–p53 interaction. Error bars indicate s.d. from two individual experiments. (b) Percentage of Annexin V-positive cells of control- or shCITED2-transduced MOLM-13 cells after incubation with indicated concentration of the inhibitor Nutlin3a for 4 h. n=2; error bars indicate s.d.; *P<0.05. (c) Schematic excerpt of the AKT signaling pathway: AKT activation requires binding of AKT to second messenger molecules (PIP3) at the cell membrane which eventually leads to expression of the pro-survival gene BCL2 and increased formation of MDM2–p53 complexes leading to p53 degradation. Binding of the p53 target PHLDA3 to PIP3 interferes with that process. (d) Western blot analysis of NB4 cells for p-AKT expression after lentiviral knockdown of CITED2. (e) Q-PCR for indicated genes in shCITED2-, shp53 or shCITED2/shp53-transduced cells. Error bars indicate Q-PCR triplicates. (f) MOLM-13 cells transduced with a short hairpin construct against CITED2 in combination with a short hairpin against PHLDA3 or a lentiviral construct for overexpression of BCL2 were stained for Annexin V. FACS plots of an individual experiment (left panels) and average percentage of Annexin V-positive cells at several days after transduction (right panels) are shown. Error bars indicated s.d. (n=2); *P<0.05, ***P<0.001