A Novel Anti-Apoptotic Role for Mir-290-295 Figure 2. Microarray data and Targetscan predictions identify candidate miR-295 targets in mESCs. A. Venn diagram of microarray and target prediction data used to generate mir-295 cluster target candidates. ‘‘mir-295 cluster predicted targets’’ = predicted TargetScan targets of the miR-295 cluster; ‘‘295 KO’’ = genes that showed a 1.2 fold upregulation on mir-295 cluster loss; ‘‘Dcr KO’’ = genes that showed a 1.2 fold upregulation on Dicer loss. Only ES-expressed genes (i.e. genes with an expression of at least 16 on the wild type arrays) were considered for analysis. B. Activity of luciferase reporters of predicted targets of the mir-295 cluster were assayed in Dcr WT and Dcr KO ES cells. Luciferase reporters contain full length 3’ UTRs of predicted targets. Relative luciferase activity is the ratio of the reporter’s activity in WT ES cells and Dcr KO ES cells. doi:10.1371/journal.pgen.1002054.g002 damage induction, the upregulation of Casp2 in Dcr KO cells appears to be insufficient to generate autocleavage. Both Dcr WT and KO ES cells showed minimal Casp3 activation immediately after 5-Gy gamma-irradiation, in line with previous descriptions of a 1–2 h lag phase in its activation (Figure 4A) [25]. However, there was a notable difference in their responses 24 h after the treatment (and to a lesser extent 10 h after treatment, Figure S4A); while 10% of WT cells became apoptotic, more than 30% of the Dcr Figure 3. The pro-apoptotic genes Caspase 2 (Casp2) and Ei24 are direct targets of the mir-295 cluster. A. RT-PCR of Casp2 mRNA in Dcr KO cells. B. Western blot of Casp2 in Dcr WT and Dcr KO ES cells. 50 nM miR-295, miR-467a, Bim siRNA, Casp2 siRNA and Ei24 siRNA were transfected into Dcr KO ES cells, and Casp2 protein expression was assayed 24 hours later. miR-467a shares the same hexamer seed with miR-295, and Bim siRNA and Ei24 siRNA served as negative controls. C. Luciferase reporters with a full length Casp2 3’ UTR or Ei24 3’ UTR, as well as their seed mutant versions were assayed by comparing their activities in Dcr WT and Dcr KO ES cells. Casp2:4M has all 4 AAGUGC seed binding sites mutated, and Ei24:1M has its single AAGUGC seed binding site mutated. 20 nM miR-295 was transfected into Dcr KO ES cells and the relative activity measured versus non- transfected cells to test if the repression of luciferase reporters is specifically due to AAGUGC miRNAs. D. Casp2 luciferase reporters bearing different combinations of AAGUGC seed binding site mutations were tested in Dcr WT and Dcr KO ES cells. Casp2:2, 2nd AAGUGC binding site was mutated; Casp2:3,4, 3rd and 4th binding sites were mutated; Casp2: 2,3,4, 2nd, 3rd, and 4th binding sites were mutated; Casp2:1,2, 1st and 2nd binding sites were mutated; Casp2: 1,2,3,4, all binding sites were mutated. n$3 for all experiments, and results are shown as mean 6 S.E.M. (standard error of the mean). P-values were obtained by t-tests, * denotes p # 0.05, and ** denotes p # 0.01. doi:10.1371/journal.pgen.1002054.g003 PLoS Genetics | www.plosgenetics.org 4 May 2011 | Volume 7 | Issue 5 | e1002054