Figure 1. Direct recognition and validation of miR-34a target genes using a luciferase assay and time-course overexpression assays of miR-34a in MB Daoy and D283-MED cell lines.

A. Top: Representative 3’-UTR diagram showing the predicted miR-34a binding sites in individual 3’-UTRs. Bottom left: Luciferase assay on Daoy cells co-transfected with individual 3’-UTR reporter constructs, the pGL3 control vector, and wild-type or seed-mutated miR-34a. The relative luciferase activities at 24 h from transfection are given, as normalized against renilla luciferase activity, and representative of six independent experiments, each performed in triplicate. The amount of transfected plasmid DNA was maintained constant by adding empty vector. *p<0.05. Bottom right: The same experimental procedures were repeated on Daoy cells using a Dll1 3’-UTR construct with mutations within the miR-34a binding site as the reporter. B. Representative Western blot time-courses for Daoy (B) and D283-MED (D) cells transfected with miR-34a, using a panel of antibodies against: Dll1, NICD1, Hey1, NICD2, Hes1 and β-Actin. C. Luciferase assay on Daoy cells co-transfected with the CBF1/RBPj-k reporter construct, the pGL3 control vector, and the wild-type or seed-mutated miR-34a. Luciferase activity at 14 h from transfection, was normalized against renilla luciferase activity. Data are representative of six independent experiments, each carried out in triplicate. The amount of transfected plasmid DNA was maintained constant by adding empty vector. *:p<0.05 D. Representative Western blot for Daoy cells 10 h from transfection with wild-type or seed-mutated-miR-34a, or at 72 h from treatment with SNALPs carrying miR-34a or SNALP-scrambled, using an anti-Dll1 antibody. Non-transfected Daoy cells were used as control. E. Representative Western blot as for (B) on D283-MED cells transfected with miR-34a F. Representative Western blot as for (B) for stable miR-34a clones 1 and 2, a stable empty vector clone, and wild-type Daoy cells.