HRG-β1-induces cancer cell migration and invasion through Smad2 activation in SK-BR-3 (a, c) and MCF7 (b, d) cells. (a, b) The motility of each cell type was assessed by wound healing assays. A scratch was made across confluent monolayers using a plastic tip and the cells were then pretreated with 10 μM of LY294002 and PD169316 or SB203580 prior to stimulation with HRG-β1. After 24 h of incubation, the migrated cells were monitored using a light microscope. Data were analyzed as percentages of the control cells in three independent experiments. *P < 0.05, significant difference. (c, d) A matrigel invasion assay was used to quantify cell invasion. After 24 h of transfection, the cells were seeded into upper chambers and incubated for 48 h in the presence of 25 ng/ml of HRG-β1. Then, the cells that invaded into the lower surface were photographed under a light microscope, with × 200 magnification. Data were analyzed as the percentage of the control of the three independent experiments. *P < 0.05 and **P < 0.01 were considered significant.