Fig. 4. Rac promotes cell locomotion of MEK1-dissociated cells. (A) Cells were microinjected with 100 ng/µl of the indicated constructs, incubated for 18 hours and then labelled for MEK1 expression. Note that MEK1SSDD-expressing cells retain the round morphology of epithelial NBT-II cells, whereas coexpression of MEK1SSDD and Rac1V12 causes cell spreading and elongation. (B) Cells were microinjected with both 100 ng/µl of the indicated constructs and 25 ng/µl of EGFP encoding construct. After 4 hours of incubation, cells were placed on a Leica motorized microscope connected to a computer using the Metamorph software, and cell motility was recorded by time-lapse videomicroscopy. Velocities were calculated by tracking EGFP-positive cells. Results are expressed as the mean of at least three independent experiments, where at least 40 EGFP-positive cells were recorded±s.e.m. (C) Cells were transfected by the PEI method with the indicated constructs together with the EGPF construct at a 1:4 ratio. 24 hours later, cells were trypsined and re-plated under sparse conditions. Cell migration was recorded 10 hours later as described above. At least three independent experiments were done, and the velocities were averaged for at least 40 cells.