Fig. 3.

Luciferase assays validate multiple Bcl-2 family members as targets of the miR-29 family. A. Bioinformatics search suggests miR-29 could target 5 members of the BCL-2 family. The seed sequences of BAK1, BBC3, BMF, BCL2L2 and MCL1 3′UTRs targeted by miR-29 are highly conserved across species (from TargetScan). B. Dual luciferase activity assays using co-transfection with a plasmid containing luciferase followed by the 3′UTRs (WT) of BCL-2 family mRNAs and plasmids encoding either pri-miR-29 wild type or pri-miR-29 with seed mutations (SM) demonstrate that miR-29ab, but not miR-29c, recognizes the 3′UTRs of all of these five BCL-2 family members, but of Bcl2L1, which is not a predicted target. C. The same assay performed with the wild type 3′UTRs or their seed mutants (SM) of the five BCL-2 family members shows that miR-29ab reduces luciferase activity, validating that miR-29ab targets all of them. Assays were performed 3 times in triplicate. *P<0.01 compared to the miR-29ab-SM or 3′UTRs-SM group (the data shown for BBC3 data comes from Ouyang et al. (2013c); the additional data is in press in Ouyang et al. (2013d)).