4 DNA damage response regulates RAG1/2 expression through ATM-FOXO1 | 85 fetal calf serum and 50 mM β-mercaptoethanol. HEK293 cells were obtained from the ATCC and maintained in DMEM (Gibco Life Technologies) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal calf serum. The following small molecule inhibitors/activators were used: STI571 (Imatinib methanesulfonate, LC Laboratories, Woburn, MA), GSK690693 (Selleckchem, Houston, TX), CAL101 (Idelalisib, Selleckchem), KU55933 (ATM-kinase inhibitor, Selleckchem), NCS (Neocarzinostatin, Sigma-Aldrich, Zwijndrecht, The Netherlands), Q-VD-OPH (Sigma-Aldrich), AKTi VIII (AKT inhibitor VIII, EMD Millipore, Billerica, MA). To induce RAG1/2 expression and Igl recombination, BV173, SUP-B15, and A70 were treated overnight with 5 mM STI571, as previously established20,21. Primary mouse bone marrow cultures ATM-deficient mice22, Jackson Laboratory stock 002753, were provided by Dr. Stephen Jones (University of Massachusetts Medical School, Worcester, MA). C57Bl/6 WT mice were housed in the animal research facility of the Academic Medical Center under specific pathogen-free conditions. Animal experiments were approved by the Animal Ethics Committee and performed in agreement with national and institutional guidelines. Bone marrow (BM) cells were harvested from 8- to 20-wk-old mice by flushing femurs and tibia. Total BM cells were cultured in six-wells plates in RPMI1640 with 2 mM L-glutamine, 100 U/ mL penicillin, 100 μg/mL streptomycin, 10% fetal calf serum, 50 mM b-mercaptoethanol, 10 ng/mL recombinant mouse IL7, and 10 ng/mL Flt3L (both from ProSpec Inc., Ness-Ziona, Israel). Culture medium and cytokines were replaced every three days. Cells were harvested after 7-10 days and analyzed by flow cytometry, typically yielding >95% B220+, IgM-, CD43+ progenitor B cells (data not shown). To induce large to small pre-B cell transition, RAG1/2 expression, and Igl recombination, cells were washed 3 times in culture medium without cytokines and placed back in six-well plates at 2x106 cells/mL in medium without cytokines (IL7 withdrawal). Induction of DNA damage DNA damage in cultured cells (2x106 cells/ml) was induced by adding 50 ng/ml NCS to the culture medium. After 2 hours the cells were harvested for respective experiments. Additionally, where so mentioned, DNA damage was induced by irradiating the cells ([137Cs], 5 Gy, 0.50 Gy/min) at a dose of 5 Gy. Following irradiation, the cells were allowed to recover for 2 hours at 37°C in an atmosphere of 5% CO2 and subsequently harvested. Western blotting and immunoprecipitation Cells (2-5x106) were washed with ice-cold PBS and lysed in ice-cold lysis buffer (10 mM TrisHCl pH 8, 150 mM NaCl, 1% NP-40, 20% glycerol, 5 mM EDTA supplemented with protease and phosphatase inhibitors (EDTA-free protease cocktail inhibitor (Roche Diagnostics, Almere, The Netherlands), 1 mM sodium-ortho-vanadate, 2 mM sodium phosphate, 1.25
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