FIGURE 3:

mTOR kinase activity is dispensable for chemotaxis. (A) Western blotting of AKT (p-S473) and S6K1 (p-T389) phosphorylation in cells pretreated with vehicle (DMSO), rapamycin (100 nM), or Torin1 (250 nM) for 30 min and stimulated with fMLP (100 nM) for indicated time points. AKT is the loading control. (B) Chemotaxis of dHL-60 cells with or without Torin1 treatment (250 nM, 30 min) in a microfluidic gradient device. Phase-contrast images of cells 10 and 480 s after fMLP stimulation. Bar, 50 μm. (C) Trajectory of dHL-60 cells migrating in the microfluidic chamber. Cell migration was recorded using time-lapse microscope, and the migration path of individual cells was analyzed and plotted using ImageJ (National Institutes of Health, Bethesda, MD). (D) Western blotting of mTOR in control and mTOR-depleted dHL-60 cells with or without rescue. mTOR-depleted cells were differentiated and transfected with WT mTOR or a kinase-dead mutant of mTOR. mTOR was depleted with shRNA-3, which targets the 3′-UTR of mTOR and thus exerts no effect on ectopically expressed mTOR. GAPDH is the loading control. (E) Both wild-type mTOR and the kinase-dead mTOR mutant rescue the migratory defects of mTOR-depleted cells revealed with the micropipette assay. Time-lapse images of representative cells for various conditions. The two images in each column show the positions of individual cells (identified with a superimposed letter) after exposure to fMLP. Bar, 10 μm. (F) Speeds of cell migration for control, mTOR-depleted, and rescued cells revealed with the micropipette assay. Values are means ± SEM (n = 18 for control, 19 for mTOR shRNA alone, 17 for mTOR shRNA plus wild-type mTOR, and 15 for mTOR shRNA plus mTOR kinase-dead mutant). Asterisks indicate that the cells differ statistically from the mTOR-depleted cells (*p < 0.001).