Figure 3.

STAT3 and Erk1/2 regulated Snail2 expression are the potential signal transducers activated by NTF1 (A) Identification of the NTF1-mediated signal transducers through blotting of the phospho-kinase array. After 24-h serum starvation, MEF^{Col1a1 4F2A Oct4-GFP} cells treated with NTF1 for 120 and 150 min were subjected to the phospho-kinase array analysis, and untreated MEFs^{Col1a1 4F2A Oct4-GFP} served as the control. The numbers labeled on the bar graph correspond to the numbers on the blot images. The upper panel shows the 120 min NTF1 treatment, whereas the lower panel displays the 150 min treatment. (B) The time-dependent effect of NTF1 treatment on the phosphorylation of STAT3 and Erk1/2. After 24-h of serum starvation, MEF^{Col1a1 4F2A Oct4-GFP} cells were subjected to a time-course experiment via treatment with 1 µg/mL NTF1 for 5, 15, 30, 60, 120, or 150 min. The NTF1-untreated MEF^{Col1a1 4F2A Oct4-GFP} cells served as the control. In the 120 min NTF1 treatment group, both phosphorylated STAT3 and Erk1/2 signals reached a plateau on the blot. (C) The dose-dependent effect of NTF1 treatment. MEF^{Col1a1 4F2A Oct4-GFP} cells were subjected to different concentrations of NTF1 (0.5, 1, and 2.5 µg/mL). Both the phosphorylated STAT3 and Erk1/2 signals reached a plateau at 1 µg/mL after 120 min of NTF1 treatment. Untreated MEF^{Col1a1 4F2A Oct4-GFP} cells served as the control. (D) The effects of NTF1 treatment in the presence of doxycycline. After 24 h of serum starvation, MEF^{Col1a1 4F2A Oct4-GFP} cells were simultaneously subjected to NTF1 and doxycycline treatment for 120 min. (E) A comparative analysis of the MET-associated genes, including Snail1, Snail2, and Thy1, revealed that Snail2 is the downstream effector of NTF1 treatment targeted by STAT3 and Erk1/2. (n = 3). ***, p < 0.0005; ns, not significant.