FIGURE 2.

mTOR-mediated regulation of PLD2 gene expression and enzymatic activity in differentiating HL-60 cells. HL-60 cells in suspension were transfected (time 0) with either scrambled siRNAs (si-Control) or siRNAs against mTOR (si-mTOR) or treated with 10 nm rapamycin or vehicle alone and immediately induced to differentiate by culturing them in fresh media in the presence of 1.25% DMSO. Total RNA for gene expression analysis by qRT-PCR or protein extracts for enzymatic activity assays in vitro was obtained at each time point, including at time 0. Results are the mean ± S.E. from three independent experiments conducted in duplicate. A, PLD2 transcript levels normalized with respect to time 0. The symbols * and # denote statistically significant (p < 0.05) differences (increases or decreases, respectively) between stimulated samples and controls, respectively. A, inset, Western blot (W.B.) of the effect of mTOR silencing on endogenous PLD2 expression as a function of increasing time. The top panel represents endogenous PLD2 protein levels using mouse anti-PLD2 monoclonal IgG antibody, 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 IgG antibodies. B, PLD lipase activity expressed as % of control (time 0) versus days of DMSO stimulation. C, mTOR mRNA expression levels normalized with respect to time 0. C, inset, Western blot of the effect of mTOR silencing on endogenous mTOR expression as a function of increasing time. The top panel represents endogenous mTOR protein levels using a rabbit anti-mTOR monoclonal IgG antibody, 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. D, total mTOR kinase activity expressed as % of control (time 0) versus days of DMSO stimulation.