miR-130a expression in AML t(8;21) and Kasumi-1 cells. The relative miR-130a expression in BM samples from 5 patients with AML with the t(8;21) chromosomal translocation, MB/PM and BC/SC cells from a healthy donor, and in Kasumi-1 cells was measured by real-time PCR with RNU6 as normalizer. Data are shown as the mean ± SD from triplicate measurements. (B) Relative expression of Smad4 mRNA in the same samples as in panel A after normalization to β-actin expression. Data are shown as mean ± SD from triplicate measurements. (C) Immunoblot of Smad4 protein expression in the 5 AML samples (AML 1-5) and Kasumi-1 cells. The exposure time was extended to get a signal in the AML samples. Immunoblot of AML5, BC/SC, MB/PM, and Kasumi-1 cells. β-actin was used a loading control. (D) Kasumi-1 cells were transiently transfected with pmiRZIP-130a (pZIP-130a) or pmiRZIP-null (pZIP-null), and Smad4 protein expression was determined by immunoblotting. The graph displays the relative expression of Smad4 normalized to β-actin expression. (E) Cell proliferation assays performed on Kasumi-1 cells transiently transfected with pmiRZIP-130a (pZIP-130a) or pmiRZIP-null (pZIP-null) 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 transfection. The mean reduction in cell number (Red. in cell no.) for TGF-β1–stimulated cells for each transfection was 38% for pZIP-130a and 7% for pZIP-null.