FIGURE 7.

Differentiation of HL-60 cells or PLD2 overexpression correlates with increased Fes kinase activity. A, HL-60 cells in suspension were induced to differentiate in fresh media containing 1.25% DMSO. Protein samples were obtained immediately after DMSO treatment (day 0) and each day post-induction of differentiation (days 1–4). Kinase assay results are the means ± S.E. from three independent experiments conducted in duplicate. Asterisks represent statistically significant (p < 0.05) differences between kinase activity values respective to day 0. B, effect of PLD2 overexpression in differentiating HL-60 cells on endogenous kinase activity associated with Fes. HL-60 cells growing in suspension were transfected with the indicated amounts of plasmid DNA encoding for fully active PLD2 and induced to differentiate in fresh media containing 1.25% DMSO. C, effect of lipase-dead mutants of PLD2 on Fes-associated kinase activity in differentiated HL-60 cells. Suspensions of HL-60 cells were nucleofected with 2.5 μg of plasmid DNA encoding for fully active PLD2 (WT) or lipase-inactive versions of PLD2 (K444R, K758R, and K444R/K758R) and induced to differentiate. Data are mean ± S.E.; asterisks represent statistically significant (p < 0.05) differences between kinase activity values respective to the values obtained in nontransfected HL-60 cells. D, Western blot (W.B.) of the effect of PLD2 overexpression. The top and middle panels represent recombinant Myc-tagged PLD2 or endogenous PLD2 protein levels, respectively, and the bottom panel is the equal loading control that shows approximately equal amounts of protein in the lysates used for electrophoresis, as ascertained with rabbit anti-actin monoclonal IgG antibodies.