Mir-29b regulates MCL-1, CXXC6, and CDK6 expression through binding with their 3 UTR. (A) Predicted site of miR-29 in the MCL-1 3′ UTR mRNA (red: nucleotides involved in the target interaction). (B) Diagram illustrating the structure of the CXXC6 and CDK6 (C) 3′ UTR. There are 4 and 3 predicted miR-29 sites in the CXXC6 and CDK6 3′ UTR, respectively. (D) A wild-type (WT) luciferase reporter plasmid was generated by fusing a fragment of the MCL-1, CXXC6, and CDK6 3′UTR encompassing the miR-29 binding site downstream of the luciferase (Luc) reporter gene. (E) Luciferase activities from the MCL-1, (F) CXXC6, and (G) CDK6 constructs were determined at 24 hours and were normalized using Renilla. The mutant plasmid was generated by deleting the miR-29 binding site. The numbers next to the mutants (ie, Mut1, Mut2) represent the numbers of predicted miR-29 binding sites deleted, starting from the 5′ end of the gene. The mutations (deletions) are additive (ie, Mut2, have both the first and second site deleted). For CXXC6 we deleted up to 3 predicted sites (Mut3 includes deletions on the 3 sites: 283-9, 400-6, and 1487-93). The data represent the average of 3 independent experiments ± SD.