Smad3 is required for TGF-β–mediated EMT, but not MMP secretion in ovarian cancer cells cultured on plastic. Cells were cultured on plastic dishes and transfected with 50 nmol/L Smad3 siRNA or control (nontargeting) siRNA and then treated with vehicle, 10 ng/mL TGF-β1, TGF-β2, or TGF-β3 for 72 h. A. Anti-Smad3 immunoblot showing down-regulation of Smad3 protein in Smad3-siRNA–transfected cells. The same blot was also reprobed with anti–β-actin as a loading control. B. Brightfield images of quiescent cells transfected with control or Smad3 siRNAs and then treated with vehicle and TGF-β ligands (top); Anti–E-cadherin immunofluorescence (white arrowheads) showing that Smad3-siRNA–transfected cells do not undergo an EMT in response to TGF-β1, TGF-β2, or TGF-β3, in contrast to control-siRNA–transfected cells (bottom); bar, 20 μm. C. Gelatin zymography of conditioned media from cells transfected with 50 nmol/L Smad3 or control (nontargeting) siRNA showing effects on MMP secretion.