Figure 7
Effect of dominant negative (dn) and constitutively active (ca) RhoA and Rac1 mutants on the Na+–H+ exchanger (NHE)1-dependent stimulationof cell motility, invasive capacity and cell shape by serum deprivation. To examine the effect of RhoA and Rac1 on serum deprivation-induced motility and invasive ability of the cells, MDA-MB-435 cell monolayers were left in fresh whole growth medium (nondeprived [ND]) or were serum deprived (D) after having been transfected with dn or ca mutants of RhoA (stippled bars) or Rac1 (stripped bars), or empty vector (empty bars) for 1 day. (a) For motility, cells were then trypsinized, centrifuged and resuspended in serum-free medium with or without 2 μmol/l 5-(N, N-dimethyl)-amiloride (DMA). Cells (20,000) were added to the upper chamber of a Boyden Chamber, in which the lower chamber containing medium plus 1% serum was separated by a collagen I coated filter with 8 μm pores. After 4 hours of incubation, filters were removed, processed as described in the Methods section, and the cells that had traversed the filter were photomicrographed (20×). The bar is 10 μm. Circles are the 8 μm pores of of the filter. (b) As described in the Methods section, the number of cells that tranversed the collagen I coated filter were counted in photomicrographs taken at 100×. Values are expressed as mean ± standard error. n = 4; **P < 0.001 versus nondeprived cells. For invasion, cells were metabolically loaded with 3H-thymidine for the 24 hours prior to the experiment, MDA-MB-435 cells were trypinsized, centrifuged and resuspended in serum-free medium, and 30,000 cells were added to the confluent MCF-10A monolayer in each well of a 96 well culture plate. After 8 hours incubation, noninvaded cells were removed and (c) the monolayer was photographed at 10× or (d) the number of invaded cells determined as described in the Methods section by counting in a Packard TopCount NXT® microplate scintillation counter. Values are expressed as mean ± standard error. n = 16; **P < 0.001 versus nondeprived cells.