Research Paper Volume 16, Issue 7 pp 6588—6612

Figure 6. Inhibition of Smad signaling downstream of TGF-β abrogated TGF-β-induced cytostasis and EMT and strengthened MAPK signaling in LPCs. (A) Western blotting was used to measure the expression of Smad4 in WB-shSmad4, LE-shSmad4, and control cells (WB-shScramble or LE-shcramble cells). GAPDH was used as a loading control. (B) CCK-8 assays of WB-shSmad4, LE-shSmad4 and control cells before and after treatment with TGF-β (10 ng/ml, 3d). OD values were normalized to those of the control groups. (C) WB-shSmad4, LE-shSmad4, and control cells were treated with TGF-β (10 ng/ml, 3d) as indicated, and Western blot analyses were carried out with antibodies against E-cadherin and vimentin. GAPDH was used as a loading control. (D) WB-shSmad4, LE-shSmad4 and control cells were treated with TGF-β (10 ng/ml) for 3 days and then subjected to phalloidin staining for F-actin (red). DAPI (blue) was used to stain the cell nuclei. The white arrows indicate F-actin rearrangements. Scale bar, 25 μm. (E) Cell motility analyses of WB-shSmad4, LE-shSmad4 and control cells treated with TGF-β. The average number of migrated cells per field is shown. (F) Western blot analyses of Smad4, p-ERK, ERK, p-p38 MAPK, p38 MAPK, p-c-Jun and c-Jun expression in WB-shSmad4, LE-shSmad4, and control cells. GAPDH was used as a loading control. WB-shSmad4, LE-shSmad4 and control cells were treated with TGF-β (10 ng/ml) for 1 h or 3 h, respectively. These experiments were repeated three times. One-way ANOVA was used for statistical analysis. The data are presented as the mean ± S.E.M. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001.