Figure 3.

Verteporfin treatment impairs TGF-β–induced Smad2/3 signaling via reducing the nuclear accumulation and total levels of Smad2/3 protein. (A) NRK49F renal fibroblasts grown in fibronectin–coated plastic dishes were treated with and without 250 nmol/L verteporfin for 12 hours and stimulated with or without TGF-β for 30 minutes. Lysates were immunoblotted for phospho-Smad2, phospho-Smad3, Smad2, or Smad3. Blots shown are representative of three replicates. (B) NRK49F fibroblasts grown on fibronectin-coated coverslips were treated with or without 250 nmol/L verteporfin for a total of 12 hours and 10 ng/ml TGF-β for the indicated times. Cells were stained with an anti-Smad2 antibody (red) and counterstained with DAPI (blue) to identify nuclei. Representative images demonstrate not only reduced nuclear accumulation of Smad2 beginning early post–TGF-β (0.5–1 hours) but also, diminished whole–cell Smad2 levels at later time points (2, 4, and 8 hours). Scale bar, 15 μm. (C) The percentage of cells with predominantly nuclear Smad2 staining was quantified at each time point. A minimum of 50 cells was counted per replicate (four replicates per condition). One-way ANOVA with post hoc Fisher least significant difference was performed. *P<0.05. In D, NRK49F fibroblasts grown on fibronectin-coated plastic were treated with 250 nmol/L verteporfin for a total of 12 hours and 10 ng/ml TGF-β for the indicated times. Verteporfin treatment resulted in a dramatic reduction in Smad2 and Smad3 post–TGF-β, a setting in which Smad2 and Smad3 are primarily in their activated state. P-Smad2, phospho-Smad2; P-Smad3, phospho-Smad3; VP, verteporfin.