miR-292 and exposed to a phosphoimager before scanning. Prior to hybridizing with a different probe, membranes were stripped by incubating the membrane in boiling 0.1% SDS for 30 minutes and loss of signal was confirmed prior to rehybridization. Western Blot analysis 24 hours after transfection with short RNAs, Dicer12/2, Dicerflox/flox, miR-290-2952/2, or miR-290-295flox/flox cells were lysed in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, in pH 7.4 PBS) containing protease inhibitors. 30–50 mg lysate was loaded onto 8–12% Bis-Tris gels (Invitrogen) and wet- transferred at 4uC to Westran PVDF membranes for 2 h at 70V. After 1 h blocking at room temperature in 5% milk-TBST, membranes were probed overnight at 4uC with 1:2000 mouse anti-vinculin (Santa Cruz Biotechnology) or 1:200 rat anti-Caspase 2 (Millipore, 10C6). After 2610 min. TBST washes, membranes were probed for 1 h at room temperature with 1:2000 corre- sponding hRP-conjugated secondary, washed an additional 2610 min. in TBST, and visualized using Western Lightning Plus ECL (PerkinElmer). RT-PCR Trizol (Qiagen) was used to extract RNA from Dicer1flox/flox and Dicer12/2 cells. A Superscript III kit (Invitrogen) was used to reverse transcribe 1 mg RNA following DNAse treatment with the Turbo-DNA free kit (Ambion), and real time PCR was performed with the primer sequences listed, using beta actin for normalization. Transfection and Casp3 assays 24 hours before transfection 2e5 mESC cells were plated/well of gelatinized 12-well plates. Cells were transfected with 4 ml Lipofectamine 2000 (Invitrogen), 0.2 mg pCAGGS-mCherry plas- mid, 1.4 mg of pWS, and 50 nM of siRNA in 600 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 exposed to 5-Gy gamma radiation or 100 nM doxorubicin. Immediately after exposure, one plate of cells were trypsinized and fixed with 16 BD Perm buffer. Cells were stained with Rabbit Anti-Casp3 antibody (BD Biosciences) at 1:100 for 20 min. at room temperature. Following washing, cells were incubated with Alexa-488-conjugated secondary antibody (diluted 1:250) (Invitrogen) for 60 min. at room temperature, washed, and resuspended in BD FACS buffer containing 1:5000 Hoechst stain. 24 hours after the treatment, another plate of cells was trypsinized and treated with the same protocol for FACS analysis. Casp3 assays were also performed on Dcr KO and WT mESCs without transfection. 24 hours before collecting cells for the 0 h time point for Casp3 assay, 2e5 mESC were plated/well of gelatinized 6-well plates. In the context of genotoxic stress, 4e5 mESCs were plated/well of gelatinized 6-well plates. 24 hours after plating, cells were treated with 5-Gy radiation or 100 nM doxorubicin. Casp3 assays were performed at 0 h and 24 h after the treatment following the same protocol described above. Annexin V assays 4e5 mESCs were plated/well of gelatinized 6-well plates. 24 h after plating, cells were exposed to 100 nM doxorubicin. Cells were trypsinized 0 h and 24 h after the treatment for Annexin V detection, following Annexin V-FITC apoptosis detection kit (BD Biosciences). Microarray analysis Microarray analysis was performed 5 days following transfection of Dicerflox/flox wild-type cells with either GFP alone or GFP and A Novel Anti-Apoptotic Role for Mir-290-295 Cre recombinase, and data were analyzed using biological triplicates. Microarrays for the mir-295 cluster deletion were performed on two deletion and two wild-type lines independently derived. Spot replicates were condensed using geometric means. The log fold change (LFC) value for Dcr WT/Dcr KO was defined as the difference between the mean log expression in Dcr WT cells and the mean log expression in Dcr KO cells. The conserved set of targets were downloaded from TargetScan- Mouse5.1 website (http://www.targetscan.org/mmu_50/). To identify targets predicted for the AAGUGC seed family, we looked at all miRNAs that contain AAGUGC in their seed region. More specifically, they include ‘‘miR-291b-3p/519a/519b-3p/ 519c-3p’’, ‘‘miR-290-3p/292-3p/467a’’, ‘‘miR-467cd’’, ‘‘miR- 106/302’’, and ‘‘miR-467b’’. We excluded all the targets of ‘‘miR-302ac/520f’’, as well as T1A 7mer targets of ‘‘miR-467b’’, as they do not contain the 6-mer match to AAGUGC. Targets with top 10% of branch length scores were considered ‘‘conserved’’. Gene Ontology and Pathway analysis Gene Set Analysis Toolkit (http://bioinfo.vanderbilt.edu/ webgestalt/) was used to perform GO analysis. Targets and controls were generated as described in the text. Network data were analyzed through the use of Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com). Ingenuity Pathway Analysis (IPA) was performed on the set of validated miR-295 targets to identify the most strongly associated canonical pathways. Statistical analyses All test statistics were calculated using R (http://www.r-project. org). The Wilcoxon rank sum test was used because it does not assume normality of the underlying distributions. T-tests and Kolmogorov–Smirnov (KS) test using these data gave generally similar results. Supporting Information Figure S1 Microarray data from polyclonal acute dicer deletion samples show better inter-sample correlation than data from clonal chronic dicer deletion lines. Biological triplicates were obtained for both 5 days post Dicer deletion (Acute 1-3) and over 1 month following Dicer deletion (Chronic 1-3), after which samples were normalized together. Pearson correlations for all pairwise compa- risons within the two conditions are shown here. (PDF) Figure S2 Repression of predicted targets of the miR-295 cluster in Dcr WT and Dcr KO ES cells. Activity of luciferase reporters of predicted targets were assayed in WT, Dcr KO ES cells, as well as in Dcr KO ES cells after over expression of 20 nM miR-295. n$2, and results are shown as mean 6 S.E.M. (PDF) Figure S3 Comparison of WT and KO cells’ apoptosis response to stress. A. The percentage of cells expressing cleaved Caspase 3 (Casp3) in WT and Dcr KO ES cells under normal culturing conditions (0 and 24 hours after plating). B. The percentage of cells expressing cleaved Caspase 3 (Casp3) in 295 WT and 295 KO ES cells under normal culturing conditions (0 and 24 hours after plating). Cleaved Casp3 was assayed by flow cytometry, and was used to estimate apoptosis response. Apoptosis of KO cells is shown in black bars, and that of WT cells is shown in white bars. C. Annexin V positive cells were assayed by flow cytometry immediately or 24 h after exposure to 100 nM doxorubicin. n = 2, and results are shown as mean 6 S.E.M. (standard error of the PLoS Genetics | www.plosgenetics.org 9 May 2011 | Volume 7 | Issue 5 | e1002054