HRG-β1 induces expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.