mean). D. Northern analysis for miR-295 in Dcr KO ES cells, WT ES cells, and WT ES cells 6 hours after 2 mM doxorubicin treatment. (PDF) Figure S4 Time course of WT and KO cells’ apoptosis response to stress. A. WT and Dcr KO ES cells were treated with 5-Gy radiation. Casp3 activity was assayed 0 h, 10 h, and 24 h after the treatment, and the differences in apoptosis rate (between 10 h and 0 h, and between 24 h and 0 h) are shown. Cleaved Casp3 was assayed by flow cytometry, and was used to estimate apoptosis response. Apoptosis rate of Dcr KO cells is shown in black bars, and that of WT cells is shown in white bars. n = 2, and results are shown as mean 6 S.E.M. B. WT and Dcr KO cells were treated with 2 doses of doxorubicin (200 nM and 300 nM). Casp3 activity was assayed 0 h, 10 h, and 24 h after the treatment, and the differences in apoptosis rate (between 10 h and 0 h, and between 24 h and 0 h) are shown. n = 1 with 3 technical replicates, and results are shown as mean 6 standard deviation. (PDF) Figure S5 Analysis of Caspase 2 and Caspase 3 cleavage upon DNA damage induction. A. Dcr KO cells treated with 2 mM doxorubicin for 6 hours. Blot was probed with an antibody specific for cleaved Caspase 3 and for its target, Nanog, before and after treatment. B. Dcr KO and Dcr WT cells following 2 mM doxorubicin treatment for 6 hours. Intact Caspase 2 (49 kD) and cleaved Caspase 2 (35 kD) are both shown. (PDF) Figure S6 Raw data of Dcr KO and WT cells’ apoptosis response after treatment of 5-Gy radiation or 100 nM doxorubi- cin. A. Dcr KO cells were treated with 5-Gy radiation 24 hours after transfection of 50 nM miR-295 or miR-290-3p. Caspase 3 activity was assayed 0 and 24 h after the treatment. Transfection of seed mutants and control siRNAs (50 nM) into Dcr KO cells, and overexpression of control siRNAs (50 nM) into WT cells served as controls. B. Dcr KO cells were treated with 5-Gy radiation 24 hours after transfection of 50 nM siRNAs against Bim, Casp2, and Ei24. Caspase-3 activity was assayed 0 and 24 h after the treatment. C,D. A similar series of experiments was performed in Dcr WT and Dcr KO cells using 100 nM doxorubicin. n $ 3 for all experiments. Results are shown as mean 6 S.E.M. (standard error of the mean). (PDF) References 1. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, et al. (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 27: 91–105. 2. Nielsen CB, Shomron N, Sandberg R, Hornstein E, Kitzman J, et al. (2007) Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. RNA 13: 1894–1910. 3. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, et al. (2008) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40: 499–507. 4. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448: 318–324. 5. Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, et al. (2005) Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev 19: 489–501. 6. Murchison EP, Partridge JF, Tam OH, Cheloufi S, Hannon GJ (2005) Characterization of Dicer-deficient murine embryonic stem cells. Proc Natl Acad Sci U S A 102: 12135–12140. 7. Leung AGY, ArjunBhutkar, Grace XZheng, Andrew DBosson, Cydney BNielsen, Phillip ASharp (2010) Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs. Nat Struct Mol Bio. A Novel Anti-Apoptotic Role for Mir-290-295 Figure S7 Raw data of 295 KO and 295 WT cells’ apoptosis response after treatment of 5-Gy radiation or 100 nM doxorubi- cin. A. 295 KO cells were treated with 5-Gy radiation 24 hours after transfection of 50 nM of miR-295 or miR-290-3p. Caspase 3 activity was assayed 0 and 24 h after the treatment. Transfection of seed mutants and control siRNAs (50 nM) into 295 KO cells, and overexpression of control siRNAs (50 nM) into 295 WT cells served as controls. B. 295 KO cells were treated with 5-Gy radiation 24 hours after transfection of 50 nM siRNAs against Bim, Casp2, and Ei24. Caspase-3 activity was assayed 0 and 24 h after the treatment. C,D. A similar series of experiments was performed in 295 WT and 295 KO cells using 100 nM doxorubicin. n $3 for all experiments. Results are shown as mean 6 S.E.M. (standard error of the mean). (PDF) Table S1 Sequences and expression level of the mir-295 cluster in ES cells. The 6-mer seed is highlighted in bold. The cloning statistics were taken from previously published studies [7,8,9]. (PDF) Table S2 Sequences and expression level of the mir-302, mir- 467, and mir-17-92 clusters in ES cells. The 6-mer seed is highlighted in bold. The cloning statistics were taken from previously published studies [7,8,9]. (PDF) Predicted targets of the mir-295 cluster. Oligos and siRNAs used in all experiments. Table S3 (PDF) Table S4 (PDF) Acknowledgments We thank J. R. Neilson and A. Seila for experimental assistance and reagents, and members of the Jaenisch, Burge, and Sharp laboratories for helpful discussions. Author Contributions Conceived and designed the experiments: GXYZ AR. Performed the experiments: GXYZ AR JMC. Analyzed the data: GXYZ AR. Contributed reagents/materials/analysis tools: LAM OK LMD RJ CBB. Wrote the paper: GXYZ AR PAS. 8. Ciaudo C, Servant N, Cognat V, Sarazin A, Kieffer E, et al. (2009) Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation. PLoS Genet 5: e1000620. doi:10.1371/journal.pgen.1000620. 9. Babiarz JE, Ruby JG, Wang Y, Bartel DP, Blelloch R (2008) Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev 22: 2773–2785. 10. Benetti R, Gonzalo S, Jaco I, Munoz P, Gonzalez S, et al. (2008) A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases. Nat Struct Mol Biol 15: 268–279. 11. Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F, et al. (2008) MicroRNAs control de novo DNA methylation through regulation of transcrip- tional repressors in mouse embryonic stem cells. Nat Struct Mol Biol 15: 259–267. 12. Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, et al. (2008) Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat Genet 40: 1478–1483. 13. Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ (2005) The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc Natl Acad Sci U S A 102: 10898–10903. 14. Calabrese JM, Sharp PA (2006) Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells. RNA 12: 2092–2102. PLoS Genetics | www.plosgenetics.org 10 May 2011 | Volume 7 | Issue 5 | e1002054