TGF-β1 sensitivity is restored by antisense RNA against miRNA-130a or expression of a Smad4 mRNA lacking miR-130a–binding sites. (A) 2 32Dcl3 clones stably transfected with pmiRZIP-130a (pZIP-130a–cl 1 and pZIP-130a–cl 2) or pmiRZIP-null (pZIP-null–cl 1and pZIP-null–cl 2) were tested for Smad4 expression by immunoblotting. The graph shows the relative expression of Smad4 normalized to β-actin expression. (B) Cell proliferation assays were performed with pZIP-null–cl 1, pZIP-null–cl 2, pZIP-130a–cl 1, and pZIP-130a–cl 2 in the presence or absence of human recombinant TGF-β1 (20nM). The mean reduction in cell number (Red. in cell no.) for TGF-β1–stimulated cells for each clone was 36% for pZIP-null–cl 1, 39% for pZIP-null–cl 2, 54% for pZIP-130a–cl 1, and 56% for pZIP-130a–cl 2 (the values are based on 3 independent growth experiments). (C) The 4 clones stably transfected with pEGP-miR-130a (Cl 3, Cl 6, and Cl 7) and pEGP-null (null) were stably transfected with a plasmid expressing Smad4 mRNA with a 3′-UTR lacking the miR-130a-binding sites (CDS). The immunoblot shows Smad4 protein expression in the clones Cl 3 + CDS, Cl 6 + CDS, Cl 7 + CDS, and null + CDS. The graph shows the relative expression of Smad4 normalized to β-actin expression. (D) Cell proliferation assays were performed with the clones Cl 3 + CDS, Cl 6 + CDS, Cl 7 + CDS, and null + CDS in the presence or absence of human recombinant TGF-β1 (20nM). The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-β1–stimulated cells was recalculated accordingly. The mean reduction in cell number (Red. in cell no.) for TGF-β1–stimulated cells for each clone was 39% for Cl 3 + CDS, 44% for Cl 6 + CDS, 37% for Cl 7 + CDS, and 42% for null + CDS (the values are based on 3 independent growth experiments).