FIGURE 7:

Reactive oxygen species are necessary for enhanced contractility and deciliation. (A, B) LLC-PK1 cells were grown to confluence and treated with serum-free medium (control) or LCM plus TGFβ for 48 h. Cells were then exposed to NBT for 45 min and processed as described in Materials and Methods. Reduced NBT (formazan) particles were first visualized by light microscopy (A) and then extracted, solubilized, and quantified (B; n = 5). (C) LLC-PK1 cells were treated as in A in the presence of dimethyl sulfoxide (DMSO) or 600 μM apocynin and processed for Western blotting for the indicated proteins. (D) Confluent LLC-PK1 cells were treated with LCM plus TGFβ for 48 h in the presence of DMSO or 600 μM apocynin and stained for Ac-tub. (E) Ciliation for B was quantified (n = 3, ∼150 cells/experiment). (F, G) LLC-PK1 cells were transfected at 60% confluence with nonrelated or Smad3 siRNA and, upon reaching confluence, treated as indicated for 48 h and processed for Western blotting for the indicated proteins. (H) Densitometric quantification of Nox4 expression in control and Smad3-depleted cells upon LCM plus TGFβ treatment, as shown in E (n = 6).