study delineating ATM function in NSCs may provide useful insight for development of stem cell-based novel therapies in human patients with A-T or with other genetic deficiencies that lead to neurodegeneration. Transplantation of normal Atm+/+ NSCs into Atm-/- mouse brains may thus promote a return to normal levels of Bmi-1 in the Atm-/- NSCs. In future studies, we will develop a reliable NSC transplantation system to ask two questions. First, is Atm+/+ NSC transplantation into the cerebella of Atm-/- mice followed by a functional recovery of Atm-/- NSC and restore the normal cerebella architecture? Second, can Atm+/+ NSC transplantation alter p38 signaling and gene expression abnormalities in the NSCs of Atm-/- mice? Materials and Methods Mice The Atm-/- mice were originally generated by Dr. C. Barlow. They were purchased from the Jackson Laboratory (Bar Harbor, ME). We genotyped offspring of Atm+/– breeding pairs by real-time polymerase chain reaction–based assays of mouse tail DNA. Littermates were used as controls in all experiments. Animal care was in accordance with The University of Texas MD Anderson Cancer Center guidelines for animal experiments. NSC isolation and neurosphere culture Neurospheres were obtained from P1 pup SVZ, and were maintained in culture essentially as reported [37]. The walls of the lateral ventricles were removed and enzymatically dissociated in HBSS buffer (5 mM KCl, 124 mM NaCl, 3.2 mM MgCl2, 100 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose) contain- ing 1 mg/ml trypsin at 37uC for 10 minutes. The tissue was centrifuged at 750 rpm for 5 minutes in soybean trypsin inhibitor. The dispersed cells were then resuspended in HBSS buffer containing 0.7 mg/ml RNase free DNase and enzymatically disaggregated for 5 minutes at room temperature. The dissociated cells were centrifuged, resuspended in Neural Basal Medium (NBM) containing 20 ng/ml EGF, 10 ng/ml FGF, 2 mM glutamine, antibiotics (100 units/ml penicillin and 100 g/ml streptomycin), and 0.125 mg/ml fungizone, and maintained in an atmosphere of 5% CO2, at 37uC. After 1–2 days of incubation, the cells formed neurospheres. Subcultures were prepared every 4–5 days by centrifugation of the neurospheres and dissociation of the cells in 1 ml of trypsin; then, the single-cell suspensions were replated in new culture dishes in fresh medium, to obtain new neurospheres. Experiments were performed with cultured cells between passages 2 and 5. GSCs were developed by Dr. F. Lang (MD Anderson Cancer Center) and were kindly provided by Dr. O. Bogler (MD Anderson Cancer Center). GSCs culture condition was same with Atm+/+ and Atm-/- NSCs as described above. Chemical reagents The PI3K inhibitor LY294001 (10 mM), the p38 MAPK inhibitor SB203580 (10 mM), the ERK inhibitor PD98059 (10 mM), MG132 (10 mM), and Okadaic acid (10 mM), were purchased from BD Biosciences. Lambda phosphatase was purchased from New England Biolabs. Transcription inhibitor cycloheximide (50 mg/ml) was purchased from Sigma-Aldrich. Generation of Bmi-1 over-expressing Atm+/+ and Atm-/- neural stem cells Bmi-1 bearing retrovirus was made using a mouse stem cell virus (MSCV) vector MSCV-GFP-Bmi-1 (kindly provided by Dr. Morrison, University of Michigan), which contains an internal ribosome entry site (IRES) followed by GFP [14]. Preparation of the ROS Accelerates Proteasomal Degradation of Bmi-1 virus was carried out by transfection of 293T packaging cells with the MSCV-GFP-Bmi-1 vector. 293T cells were allowed to produce virus for 24–48 hours, then cell supernatants were collected, 0.2 micron filtered, and stored at 280uC or used fresh to infect cultured NSCs. To generate Bmi-1 over-expressing Atm+/+ and Atm-/- NSCs, neonatal subventricular zone (SVZ) tissue from wild type or Atm deficient mice was dissected, dissociated, and cultured as described above. NSCs were plated at 20,000 cells per well of a six-well plate. After 24–48 hours, virus-containing supernatant was added to NSC cultures for 16 hours. The culture medium was then replaced for 24 hours, and cells were trypsinized and re-plated for tests of proliferative capacity. Before proliferative capacity assays, MSCV- GFP-Bmi-1 expression in cells was assessed by fluorescence microscopy. Cell proliferation assays After trypsinizing neurospheres, single neural stem cells were resuspended in NBM containing 20 nM EGF and 20 nM FGF. The cells were then seeded into 96-well plates at a density of 102 cells per well, to allow cell communication by paracrine/autocrine factors for physiological proliferation. After incubation for 48 hours, the numbers of newly formed neurospheres per well were counted, using phase contrast microscopy. To measure neurosphere sizes, images were obtained from 3 separate fields per well, and the volumes of neurospheres estimated using a microimage analysis system from Olympus. To measure total cell proliferation in neurospheres, cells in each well were trypsinized, and total cell numbers per well were counted. Immunocytochemical image analysis Neurospheres were seeded on 2– or 8– well chamber slides coated with poly-L-lysine/lamin, and then incubated for 3 hours, to facilitate adhesion. These cells, which will be referred to as ‘‘adhered neurospheres’’ in the text, maintained the properties of undifferentiated cells, as evidenced by anti-nestin staining. Adhered neurospheres also respond to H2O2 in a manner similar to the floating neurospheres. For ROS response experiments, neurospheres in monolayer cultures were treated with H2O2 in NBM for 16 hours, after which the cells were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature, and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. For staining with antibodies, the cells were preincubated for 2 hours at 37uC with 10% FBS in PBS, and then further incubated at 4uC overnight with primary antibodies. The antibody-bound cells were washed three times in PBS for 5 minutes each, and fluorescent secondary antibodies were added for 1 hour at 37uC. Nuclei were stained with 49, 6-diamidino-2- phenylindole (DAPI). The stained slides were then mounted in Slowfade (Molecular Probes). Cells were imaged under an Olympus IX2-SL microscope, equipped with a 6400 objective and an Olympus DP 70 digital camera connected to a PC computer. Protein analysis For protein measurement, cells were seeded into 10-cm dishes, and treated with drugs or H2O2. At indicated times after treatment, cells were collected and washed once with ice-cold PBS and then resuspended into lysis buffer containing 150 mM NaCl, 0.5% w/v sodium dodecyl sulfate (SDS), 0.5% v/v NP-40, 0.5% w/v sodium deoxycholate, 1 mM EGTA, and a mixture of protease inhibitors (Complete Mini tablets; Boehringer Mann- heim). For Western blotting, protein concentrations were deter- mined using a Bradford reagent (BioRad). Proteins (30 mg) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels, PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16615