ZFIN Image: Le et al., 2011, Figure 1

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Figure Caption

Figure 1 Identifying miR-125b targets in the p53 network of vertebrates.

Schematic of experimental design and workflow. (A) Bioinformatic analysis was performed on p53 network genes listed in the Ingenuity Pathways Analysis database and p53 Knowledgebase, and miR-125b binding sites predicted by the TargetScan and MicroCosm databases. (B) p53 network genes were screened for miR-125b targets by using gain- (GOF) and loss-of-function (LOF) of miR-125b in human cells, mouse cells and zebrafish embryos, as indicated by effects on gene expression using qRT-PCR. (C) p53 network genes that were positive in either the GOF or LOF screen were assayed for direct binding to miR-125b using a biotinylated microRNA pull-down method. (D) p53 network genes that were also positive in the miR-125b pull-down were finally validated as miR-125b targets by 3′ UTR luciferase reporter assays and Western blots for protein expression. (E) A model of how miR-125b regulates the p53 network across vertebrates was constructed using our combined datasets for human, mouse and zebrafish cells.

Acknowledgments

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