Estramustine Phosphate Inhibits TGF-β-Induced Mouse Macrophage Migration and Urokinase-Type Plasminogen Activator Production

Figure 4

EP inhibits TGF-β-induced Smad3 activation. (a) TGF-β 5 ng/ml was used to one-hour stimulation of RAW 264.7 cells in the presence or absence of EP 10 μM. Then, samples were subjected to the WB assay to determine Smad3 phosphorylation levels. EP reduced the capacity of TGF-β to increase phospho-Smad3 levels. (b) Nuclear phospho-Smad3 localization. Cells were treated for one hour with TGF-β 5 ng/ml in cotreatment with EP 10 μM. Then cells were subjected to the immunofluorescence assay for Smad3 (green) and nuclei DAPI stains (blue). EP reduced the TGF-β-induced pSmad3 nuclear levels as is indicated by the number of pSmad3 positive cells. (c) EP inhibits Smad3 responsive p (CAGA)₁₂-Luc construct transactivation. Transfected cells were treated with TGF-β 5 ng/ml with or without EP 10 μM for 24 h. Then, luciferase activity was determined. RLU: relative luciferase units. (d) Direct Smad3 inhibition by using SiS3 reduces TGF-β-induced cell migration, determined by the wound healing assay. (e) Smad3 inhibition decreases TGF-β-induced uPA secreted activity, determined by the zymography assay. Representative results from three independent experiments are shown. Significant difference between treatments by t-test: and .

(a)

(b)

(c)

(d)

(e)