158 | Chapter 6 Supplement Materials and methods Mouse and human v-Abl cells The v-Abl transformed mouse pre–B cell lines were generated from wild type (WT) (A70) and Rag2-/- (R2K3) mice that harbor the Em-Bcl2 transgene and were kindly provided by Dr. Craig Bassing (University of Pennsylvania School of Medicine, Philadelphia, PA). The cells were cultured in RPMI1640 (Life Technologies, Bleiswijk, The Netherlands) supplemented with 2 mM of L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS (Thermo Scientific, Waltham, MA) and 50 μM 2-mercaptoethanol (2-ME, Sigma Aldrich, Burlington, MA). The human BCR-ABL+ B-ALL cell line BV173 was obtained from DSMZ (Braunschweig, Germany) and cultured in RPMI1640 (Life Technologies, Bleiswijk, the Netherlands) supplemented with 2 mM of l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 20% FCS. The following small-molecule inhibitors/activators were used: STI571 (imatinib methanesulfonate; LC Laboratories, Woburn, MA) to induce RAG1/2 expression in v-Abl cells and Igl recombination, the cells were treated with 5 μM STI571 as previously established1,2, KU55933 (ATM kinase inhibitor; Selleckchem, Houston, TX), Nutlin-3 (non-genotoxic p53 activator3, Sigma Aldrich), Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethyl ketone; a pan-caspase inhibitor) (Promega, Madison, WI), Staurosporine (inductor of apoptosis through caspase-activation4) (Selleckchem). Mouse primary B cells ATM-deficient mice5 (Jackson Laboratory stock 002753) and TP53-deficient mice6 were provided by Dr. Stephen Jones (University of Massachusetts Medical School, Worcester, MA). RAG1-deficient VH81x IgH m chain transgenic mice were provided by Prof. Dr. Rudi Hendriks (Erasmus Medical Center, Rotterdam, the Netherlands), 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 cells were harvested from 8- to 20-wk-old mice by flushing femurs and tibia. Total bone marrow cells were cultured in six-well plates in RPMI1640 with 2 mM of L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FCS, 50 μM 2-ME, 10 ng/ml recombinant mouse IL-7, and 10 ng/ml Flt3L (both from ProSpec, Ness-Ziona, Israel). The culture medium and cytokines were replaced every 3 days. Cells were harvested after 7–10 d and analyzed by flow cytometry, typically yielding > 95% B220+IgM- progenitor cells (Suppl Figure 1A). To induce large to small pre–B cell transition, RAG1/2 expression, and Igk recombination, the cells were washed three times in culture medium without cytokines and placed back in six-well plates at 2 × 106 cells/ml in medium without cytokines (IL-7 withdrawal) (Suppl Figure 1B).
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