and transferred to polyvinylidene difluoride membranes prior to incubation with primary antibodies. Antibodies Antibodies used for Western blotting analysis were anti- phospho-Akt (Ser473), anti-Akt, anti-phospho-p38 (Th180/ Tyr182), anti-p38, anti-ubiquitinated histone H2A, anti-Akt substrate (Cell Signaling Technology); anti-p21 and anti-b-actin (Santa Cruz Biotechnology); anti-Bmi-1 (Millipore Co.). Quantitative RT-PCR Expression levels for bmi-1, p21Cip1, p16Ink4a, and p19Arf were quantified, relative to gapdh, internal RNA control, by quantitative RT-PCR. Sequences of PCR primers are listed as follows. Primers that amplified bmi-1: Mm03053308_g1. Primers that amplified p21Cip1: cdkn1a (p21): Mm00432448_m1. Primers that amplified p16Ink4a: Mm01257348_m1. Primers that amplified p19Arf were designed: sense, 59-GGGCCGCACCGGAAT-39 and antisense, 59-AAGAGCTGCTACGTGAACGT-39. Statistics For each experiment, data are presented as the mean 6 S.E. of values. Each experiment was repeated at least three times. Statistical comparisons of values for Atm+/+ vs. Atm-/- neurospheres, and for untreated control vs. treated neurospheres were made using an analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Differences were considered significant when p,0.05. Analyses of data were performed using Prism 5 Software (GraphPad Software, Inc. San Diego, CA). References 1. Allen DM, van Praag H, Ray J, Weaver Z, Winrow CJ, et al. (2000) Ataxia telangiectasia mutated is essential during adult neurogenesis. Genes Dev 15: 554–566. 2. Gage FH (2000) Mammalian neural stem cells. Science 287: 1433–1438. 3. Chen P, Peng C, Luff J, Spring K, Watters D, et al. (2003) Oxidative stress is responsible for deficient survival and dendritogenesis in purkinje neurons from ataxia-telangiectasia mutated mutant mice. J Neurosci 23: 11453–11460. 4. Liu N, Stoica G, Yan M, Scofield VL, Qiang W, et al. (2005) ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes. Lab Invest 85: 1471–1480. 5. Kim J, Wong PK (2009) Oxidative Stress Is Linked to ERK1/2-p16 Signaling- mediated Growth Defect in ATM-deficient Astrocytes. J Biol Chem 284: 14396–14404. 6. Kim J, Wong PK (2009) Loss of ATM Impairs Proliferation of Neural Stem Cells Through Oxidative Stress-Mediated p38 MAPK Signaling. Stem Cells 27: 1987–1998. 7. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, et al. (2003) Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425: 962–967. 8. Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R (2005) Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev 19: 1432–1437. 9. Li Z, Cao R, Wang M, Myers MP, Zhang Y, et al. (2006) Structure of a Bmi-1- Ring1B polycomb group ubiquitin ligase complex. J Biol Chem 281: 20643–20649. 10. Leung C, Lingbeek M, Shakhova O, Liu J, Tanger E, et al. (2004) Bmi1 is essential for cerebellar development and is overexpressed in human medullo- blastomas. Nature 428: 337–341. 11. Fasano CA, Dimos JT, Ivanova NB, Lowry N, Lemischka IR, et al. (2007) shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development. Cell Stem Cell 1: 87–99. 12. Liu J, Cao L, Chen J, Song S, Lee IH, et al. (2009) Bmi1 regulates mitochondrial function and the DNA damage response pathway. Nature 459: 387–392. 13. Chatoo W, Abdouh M, David J, Champagne MP, Ferreira J, et al. (2009) The polycomb group gene Bmi1 regulates antioxidant defenses in neurons by repressing p53 pro-oxidant activity. J Neurosci 29: 529–542. 14. He S, Iwashita T, Buchstaller J, Molofsky AV, Thomas D, et al. (2009) Bmi-1 over-expression in neural stem/progenitor cells increases proliferation and neurogenesis in culture but has little effect on these functions in vivo. Dev Biol 328: 257–272. ROS Accelerates Proteasomal Degradation of Bmi-1 Differentiation of neural stem cells Neurospheres were enzymatically dissociated as described above. The cells were seeded on to chamber slides, and then maintained in medium containing 10% FBS, without EGF and FGF for 7 days. Antibodies used for characterization of neural stem cells were anti-Map2 (Cell Signaling Technology), anti- GFAP (Santa Cruz Biotechnology). Supporting Information Bmi-1 downregulated in Atm-/- NSCs Atm+/+ Figure S1 and Atm-/- neurospheres were analyzed for phospho-p38, phos- pho-Akt, Bmi-1, and p21 by Immunofluorescence. Cells were counterstained by DAPI (49-6-Diamidino-2-phenylindole), which identifies the nuclei of the NSCs. (DOC) Acknowledgments We are grateful to Dr. Morrison for a gift of MSCV-Bmi-1 construct and thank Hilary Graham for critically reviewing this manuscript. Author Contributions Conceived and designed the experiments: JK. Performed the experiments: JK HJ. Analyzed the data: PW JK. Contributed reagents/materials/ analysis tools: JK. Wrote the paper: JK. Obtained permission for use of construct: SM. Obtained permission for use of cell line: OB. 15. Bhattacharyya J, Mihara K, Yasunaga S, Tanaka H, Hoshi M, et al. (2009) BMI-1 expression is enhanced through transcriptional and posttranscriptional regulation during the progression of chronic myeloid leukemia. Ann Hematol 88: 333–340. 16. Chen W, Sun Z, Wang XJ, Jiang T, Huang Z, et al. (2009) Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol Cell 34: 663–673. 17. Torroglosa A, Murillo-Carretero M, Romero-Grimaldi C, Matarredona ER, Campos-Caro A, et al. (2007) Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway. Stem Cells 25: 88–97. 18. Stock JK, Giadrossi S, Casanova M, Brookes E, Vidal M, et al. (2007) Ring1- mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat Cell Biol 9: 1428–1435. 19. Li Z, Cao R, Wang M, Myers MP, Zhang Y, et al. (2006) Structure of a Bmi-1- Ring1B polycomb group ubiquitin ligase complex. J Biol Chem 281: 20643–20649. 20. Zuluaga S, Alvarez-Barrientos A, Gutie´rrez-Uzquiza A, Benito M, Nebreda AR, et al. (2007) Negative regulation of Akt activity by p38alpha MAP kinase in cardiomyocytes involves membrane localization of PP2A through interaction with caveolin-1. Cell Signal 19: 62–74. 21. Westermarck J, Li SP, Kallunki T, Han J, Ka¨ha¨ri VM (2001) p38 mitogen- activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression. Mol Cell Biol 21: 2373–2383. 22. Gonzalez I, Tripathi G, Carter EJ, Cobb LJ, Salih DAM, et al. (2004) Akt2, a Novel Functional Link between p38 Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways in Myogenesis. Mol Cell Biol 24: 3607–3622. 23. Lee B, Kim CH, Moon SK (2006) Honokiol causes the p21WAF1-mediated G(1)-phase arrest of the cell cycle through inducing p38 mitogen activated protein kinase in vascular smooth muscle cells. FEBS Lett 580: 5177– 5184. 24. Soulez M, Saurin AJ, Freemont PS, Knight JC (1999) SSX and the synovial- sarcoma specific chimaeric protein SYT-SSX co-localize with the human Polycomb group complex. Oncogene 18: 2739–2746. 25. Obuse C, Yang H, Nozaki N, Goto S, Okazaki T, et al. (2004) Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase. Genes Cells 9: 105–120. PLoS ONE | www.plosone.org 11 January 2011 | Volume 6 | Issue 1 | e16615