Figure 4

Increased p53 activation in CITED2-knockdown cells is not directly mediated by CBP/p300. (a) Western blot analysis for ac-p53 (Lys382) of sorted GFP⁺ NB4 cells transduced with a control- or shCITED2-expressing construct. Cells were sorted 4 days after transduction. Numbers indicate quantification of the ac-p53 signal normalized to Actin. (b) GFP-coimmunoprecipitation (IP) experiment of CITED2:EGFP-expressing NB4 cells and western blot analysis for indicated antibodies. Red rectangle highlights the lane showing interactions between CITED2 and the indicated proteins. (c) NB4 cells were transduced and sorted as in (a). Cells were treated with indicated concentration of the CBP/p300 inhibitor C646 for 24 h before sorting. Western blot analysis for ac-p53 (left panel) and Q-PCR analysis for PHLDA3 (middle panel) and BCL2 (right panel) expression is shown. Error bars indicate s.d. of Q-PCR triplicates. (d) NB4 cells were simultaneously transduced with indicated combinations of shRNA constructs for downregulation of CITED2, EP300 and CBP. Western blot analysis for indicated antibodies of sorted cells is shown. (e) Annexin V staining of MOLM-13 cells transduced as in (d). Percentage of Annexin V-positive cells is indicated. Error bars indicate s.d. of two individual experiments; *P<0.05