FIG 5.

HIF-1α mediates HRG-induced sensitization of Rac1 activation and motility. (A) MCF-7 cells were incubated for 16 h with either HRG (10 ng/ml) or vehicle (control) in the presence of the HIF-1α inhibitor SC205346 (3 μM, added 1 h before and during HRG or vehicle incubation). Four hours 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. (B) Rac1 activation by SDF-1 was determined in MCF-7 cells transfected with either HIF-1α or nontarget control RNAi duplexes. (A and B) (Left) Representative experiments. (Right) Densitometric values of Rac1-GTP levels (means and SEM, normalized to total Rac1; n = 3), expressed as fold increase relative to vehicle-treated NTC cells (minus SDF-1). (C) MCF-7 cells subjected to either HIF-1α or NTC RNAi were treated for 16 h with either HRG (10 ng/ml) or vehicle. Motility in response to SDF-1 (100 ng/ml; 16 h) was determined in a Boyden chamber. Shown is the effect of HIF-1α RNAi on MCF-7 cell motility. (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. C, control (vehicle). **, P < 0.01; ***, P < 0.001.