A Novel Anti-Apoptotic Role for Mir-290-295 Figure 6. Ingenuity Pathway Analysis (IPA) of miR-295 targets. IPA was performed for validated targets of miR-295 from this and prior studies [10,11,12], and identified the network, ‘‘Cell Death, Cell Cycle, Cellular Function and Maintenance,’’ which centers around p53. Solid lines indicate direct interactions and dashed lines indicate indirect interactions. Validated targets of miR-295 are shown in red and computationally predicted targets are shown in orange. doi:10.1371/journal.pgen.1002054.g006 will be described in an upcoming publication (Medeiros et. al., manuscript in preparation). Oligos and siRNAs used in all the experiments See Table S4. Generation of luciferase constructs, mESC transfection, and luciferase assays MicroRNA mediated repression of each candidate gene was tested by cloning PCR amplified products corresponding to the entire 3’ UTR downstream of a pRL-CMV Renilla luciferase reporter as described previously [41]. Nucleotides 5–7 of Casp2, Bim, and Ei24 binding sites were mutated by Quickchange site- directed mutagenesis. Digests were performed using either XhoI or SalI to give the 5’ site and ApaI or NotI to give the 3’ site. Firefly luciferase (pGL3) was used as a transfection control. Data shown are summaries of three or more independent trials. 24 hours before transfection 1e5 mESC cells were plated/well of gelatinized 24-well plate. Cells were transfected with 2 ml Lipofectamine 2000 (Invitrogen), 0.1 mg of CMV-GFP plasmid (Invitrogen), 0.7 mg of pWS (carrier plasmid), and 50 nM siRNAs in 300 ml of Opti-MEM (Invitrogen). 4 hours after transfection, transfection mix was removed from cells and replaced with ESC media. 24 hours after transfection, cells were lysed with 1X Passive Lysis Buffer (Promega) and Dual luciferase was measured using Dual Luciferase reporter assay system (Promega) according to manufacturer’s instructions. Northern Blot analysis Total RNA was isolated from ES cells with or without acute Dicer deletion using Trizol (Invitrogen), following the standard protocol. Approximately 50 mg of each RNA was loaded onto a 15% denaturing MOPS gel, according to the Northern Blot protocol outlined previously [42]. Membranes were probed for PLoS Genetics | www.plosgenetics.org 8 May 2011 | Volume 7 | Issue 5 | e1002054