HRG-β1 induces nuclear colocalization of phospho-Smad2 and Snail. Immunofluorescence analyses were performed for the nuclear colocalization of phospho-Smad2 and Snail in SK-BR-3 cells. Cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 for 1 h prior to stimulation with 25 ng/ml of HRG-β1. After incubation for a further 24 h, phospho-Smad2 (red) and Snail (green) were observed under a confocal laser scanning microscope and the nuclear DNA was stained with DAPI (blue; magnification, ×200). The lower graphs show the fluorescence intensities of phospho-Smad2 and Snail as percentages compared with control cells in three independent experiments involving SK-BR-3 (left) and MCF7 (right) cells. *P < 0.05, **P < 0.01, significant difference.