The effect of knocking-down Smad4 expression with a shRNA against Smad4 mRNA. (A) Immunoblot showing Smad4 protein expression in 32Dcl3 stably transfected with a plasmid encoding a short hairpin against Smad4 (sh-Smad4) or a nonsilencing short hairpin (sh-neg), respectively. The graph shows the relative expression of Smad4 normalized to β-actin expression. (B) The relative expression level of Smad4 mRNA in the 32Dcl3 clones sh-Smad4 and sh-neg, respectively. Smad4 mRNA expression was measured by real-time PCR and normalized to β-actin expression. Data are shown as the mean ± SD from triplicate measurements. (C) Cell proliferation assays were performed with the 32Dcl3 clones sh-Smad4 and sh-neg in the presence or absence of human recombinant TGF-β1 (20nM). The graph shows the relative number of cells after 4 days when TGF-β1–stimulated cells are compared with unstimulated cells for each clone. The reduction in cell number (Red. in cell no.) for TGF-β1–stimulated cells for each clone was 19% for sh-Smad4 and 47% for sh-neg. The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-β1–stimulated cells was recalculated accordingly.