Research Paper Volume 16, Issue 7 pp 6588—6612

Figure 8. Smad3 phosphorylation affects TGF-β-induced cytostasis and EMT in LPCs. (A) Schematic representation of the phosphorylation sites in Smad3, the corresponding C-terminal region (3SA) and the corresponding linker region (EPSM). (B–D) WB-F344 and LE/6 cells were infected with adenoviruses carrying GFP (Ad-GFP), Smad3 (Ad-Smad3), or Smad3 (Ad-3SA or Ad-EPSM) at the indicated MOIs and were treated with TGF-β (10 ng/ml) for 3 1 h. Cell lysates were subjected to Western blot analyses with antibodies against the indicated phospho- and total Smad3 proteins. GAPDH was used as a loading control. (E) WB-F344 (upper panel) and LE/6 (lower panel) cells were infected with adenoviruses carrying Smad3 or its mutants, treated with TGF-β (10 ng/ml, 3d), and subjected to CCK-8 analyses. OD values were measured and compared between groups as indicated. (F) WB-F344 and LE/6 cells infected with adenoviruses carrying Smad3 or its mutants as indicated were treated with TGF-β (10 ng/ml, 3d) 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. (G) WB-F344 and LE/6 cells infected with adenoviruses carrying Smad3 or its mutants as indicated were treated with TGF-β (10 ng/ml) for 3 days, and Western blot analyses were performed with antibodies against E-cadherin and vimentin. GAPDH was used as a loading control. (H) Cell motility analyses of WB-F344 and LE/6 cells infected with adenovirus carrying Smad3 or its mutants as indicated and control cells treated with TGF-β. The average number of migrated cells per field is shown. The experiments were repeated three times. Two-tailed Student’s t-tests were used for statistical analysis. The data are presented as the mean ± S.E.M. ^*p < 0.05; ^**p < 0.01; and ^***p < 0.001.