FIG. 6.

Src-A increases velocity and directional persistence of migration on BM. (A) Src-L or Src-A cells seeded onto BM of MCF-10A cells in the presence of dimethyl sulfoxide (DMSO) solvent or of 10 μM PP2. Images were recorded 1 h after seeding. Arrowheads indicate lamellipodia in solvent-treated cells. The area covered by individual cells was quantified using NIH image software. Bars show averages of areas covered and standard deviations (n = 19 cells from four randomly selected spots). (B and C) Src-L and Src-A cells were trypsinized and kept in suspension in starvation medium for 1 h at 37°C, seeded onto regular tissue culture plastic (B) or onto the BM of MCF-10A cells (C), and lysed at the indicated time points after seeding. Wb of total cell lysates, using anti-Src2 and anti-pY416 Src antibodies. (D) MCF-10A, Src-L, or Src-A cells were seeded as described in the legend to panel C. Cells were left unstimulated or treated with EGF. Cell movements were recorded by live cell video microscopy over 2 h, tracked, and plotted. (E) The average velocity and median directionality of cells treated as described in the legend to panel D were determined. Average velocities ± standard errors of the means (SEM) were as follows: MCF-10A cells, 0.96 ± 0.043 μm/min without EGF and 1.42 ± 0.1 with EGF; Src-L cells, 1.2 ± 0.10 μm/min without EGF and 1.64 ± 0.11 μm/min with EGF; Src-A cells, 1.45 ± 0.1 μm/min without EGF and 1.95 ± 0.24 μm/min with EGF. Medians ± SEM of directionality were as follows: MCF-10A cells, 0.31 ± 0.026 without EGF and 0.64 ± 0.067 with EGF; Src-L cells, 0.46 ± 0.081 without EGF and 0.55 ± 0.033 with EGF; Src-A cells, 0.67 ± 0.057 without EGF and 0.69 ± 0.046 with EGF (n = 45 randomly selected cells from four experiments; y-axis error bars are SEM).