Figure 3

Analysis of miRNA targets. (A) Venn diagram summarizing the rationale for selecting putative miRNA targets by combining the lists generated with prediction algorithms with those generated from expression datasets (1,858 genes with a fold change <0.5). (B) Gene Ontology (GO) enrichment analysis of putative miRNA targets from the previous analysis. (C) Summary of putative targets and their corresponding miRNA matches among the miR-99b/125a/let7e and miR-132/212 clusters. (D) Luciferase assays of HeLa cells cotransfected with different luciferase reporter psiCheck2 constructs containing the 3′ UTR of putative targeted transcription factors (wild type (WT) or mutant (Mut) forms). (E) Effects on validated targets of the single transfection with miRNA power inhibitors in MOs 4 days after being stimulated with RANKL/M-CSF, as assessed by qRT-PCR and western blotting. Expression data are relative to the levels obtained for the samples transfected with control power inhibitor or antagomir (a-miR) and are normalized to the RPL38 gene. Protein data have been normalized against α-tubulin, using the sample transfected with the control power inhibitor as a reference. At the bottom, quantification of the levels of protein relative to the control for each antagomir. (F) Effects on validated targets of the double transfection with miRNA power inhibitors in MOs 4 days after being stimulated with RANKL/M-CSF, as assessed by qRT-PCR and western blotting. Data analyzed as above. Error bars correspond to standard deviation of three independent experiments; *corresponds to P-value <0.05; **means P-value <0.01; ***means P-value < 0.001.