Journal: Journal of Hematology & Oncology

Article Title: Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival, p53-dependent apoptosis and daunorubicin-induced cytotoxicity

doi: 10.1186/1756-8722-6-78

Figure Lengend Snippet: 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.

Article Snippet: Twenty to 50 μg of WCE were subjected to SDS-PAGE, transferred to nitrocellulose or PVDF membranes and immunoblotted with the following primary antibodies: CK2α-subunit rabbit antiserum raised against the (376–3919) region of human protein (kindly provided by Dr S. Sarno, University of Padua, Italy); anti-PARP, anti-STAT3 and phospho-Ser727-STAT3, anti-MCL1 (Cell Signaling, Beverly, MA); anti-SOCSs3 and anti-CDC37 (Santa Cruz Biotechnology, Santa Cruz, CA) anti-phospho-Ser13 CDC37 (Abcam); anti-caspase 3 (Calbiochem-Merck Biosciences, bad Soden, Germany); anti-p53 and CK2β (BD Biosciences, USA); βactin (Sigma-Aldrich), GAPDH (Ambion).

Techniques: Inhibition, Staining, Transfection, Plasmid Preparation, Expressing, Microscopy