FIG 2.

P-Rex1 mediates SDF-1-induced activation of Rac1 and motility in breast cancer cells. (A) BT-474, MDA-MB-361, and MCF-7 cells were subjected to either P-Rex1 or NTC RNAi and 48 h later incubated for 16 h with either HRG (10 ng/ml) or vehicle (C). Four h after HRG removal, the cells were treated with SDF-1 (100 ng/ml; 2 min), and Rac1-GTP levels were determined using a pulldown assay. Phospho-Akt, phospho-Erk, total Rac1, and P-Rex1 levels were determined by Western blotting in cell lysates. (Top) Representative experiments. (Bottom) Densitometric values of Rac1-GTP levels (means and SEM, normalized to total Rac1) expressed as fold increase relative to vehicle-treated NTC cells (minus SDF-1). (B) MCF-7 cells subjected to either P-Rex1 or NTC RNAi were treated for 16 h with either HRG (10 ng/ml) or vehicle (C). Motility in response to SDF-1 (100 ng/ml; 16 h) was determined using a Boyden chamber. (Left) Representative images. (Right) Quantification of migrating cells by contrast microscopy in 5 independent fields. The results are expressed as means and SD of triplicate measurements. Two additional experiments gave similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001.