Figure 3

CK2 controls p53 protein levels in AML cells and p53 is essential for CK2-inhibition triggered apoptosis. (A) Representative WB analysis on ML2 cells treated with DMSO 0.1% (Un) or 5 μM K27 for 6 hours and probed with an anti-p53 antibody. Graph below: representative densitometric analysis (n = 3; p < 0.05). (B) Top: representative WB analysis of total PARP levels in Saos2 osteosarcoma cells treated with CX-4945; bottom: graph summarizing annexin V staining and FACS analysis of Saos2 cells untreated (Un) or treated with increasing concentrations of CX-4945. (C) Representative WB of pro-caspase 3 and p53 levels in Saos2 cells untransfected (Un) or transfected with pCMV empty-plasmid (pCMV) or pCMV-p53 expressing plasmid and treated either with DMSO 0.1% (-) or with 10 μM CX-4945. (D-F) Graphs summarizing the annexin V staining/FACS analysis of Saos2 cells untransfected (Un) or transfected with pCMV empty (pCMV) or pCMV-p53 plasmid and treated either with DMSO 0.1% or with 15 μM CX-4945 (D) or with DMSO 0.1% or with 10 μM K27 (E) or re-transfected with scrambled or CK2-directed siRNAs (F). Data represent mean ± SD, n = 3. * indicates p < 0.05. (G) Top: graph summarizing the data of annexin V/FACS analysis on HL-60 AML cells transfected with pCMV empty (pCMV) or pCMV-p53 plasmid and treated either with DMSO 0.1% or with 5 μM CX-4945; bottom: representative WB of p53 and pro-caspase 3 protein levels. (H) Microscope analysis of Wright-Giemsa stained HL-60 cells transfected with pCMV empty vector or pCMV-p53 and treated either with vehicle (DMSO 0.1%) or CX-4945 7.5 μM. (I) Quantification of morphological changes (shrinkage, nuclear picnosis, blebbing or apoptotic bodies) observed in the conditions as in (H). In all the experiments, either βactin or GAPDH levels were determined to ensure equal protein loading.