Figure 4
CK2 activity in γδ T-ALL cells is potentiated by CD27 costimulation and promotes AKT signaling. (a) CK2α activity in the γδ T-ALL cell line, PEER (2 × 106 cells per condition), after 6 h of treatment with indicated concentrations of CX-4945. (b) CK2α activity in lysates from γδ T-ALL (PEER) cells (2 × 106 cells per condition) after 6 h of stimulation under the indicated conditions (T-test, *P<0.05; **P<0.01). (c) Western blot analysis of (phospho)proteins implicated in AKT signaling, in γδ T-ALL (PEER) cells treated for 6 h with anti-CD3 antibodies (CD3), plus soluble CD27-ligand (CD3+CD27) or plus 5 μM CX-4945 (CD3+CD27+CX). Data are representative of five independent experiments. (d) Flow cytometry analysis of apoptosis (Annexin-V+; left panel), cell cycle/DNA staining (middle panel) and intracellular Bcl-2 protein staining (right panel; values indicate mean fluorescence intensity (MFI)) of γδ T-ALL (PEER) cells treated with CX-4945 (5 μM) during the indicated times. (e, f) Western blot analysis of phospho-AKT (and calnexin loading control) (e) and cell survival after 48 h (f) of PEER cells transduced with a bicistronic retroviral DNA construct: either empty vector (LZRS) expressing only IRES followed by eGFP (LZRS-IRES-eGFP) or vector co-expressing myrPKB/AKT and eGFP (LZRS-myrPKB/AKT-IRES-eGFP) (AKThi) and treated with 3 μM CX-4945 or vehicle (T-test, *P<0.05).
