5 NF-κB and AKT signaling prevent DNA damage by suppressing RAG1/2 | 119 genes for human and mouse samples, respectively. Primer sequences are listed in SUPPL Table 1. Analysis of Jk2-RSS signal-end DNA breaks in G1 and S phase cell cycle cells Ligation-mediated PCR (LM-PCR) for Jk2-RSS signal-end DNA breaks in cell cycle phase sorted WT and RAG2−/− Abl-transformed pre-B cells was performed essentially as described earlier,23,24 with minor modifications. A detailed protocol is available in the supplemental Data. NF-kB transcription factor activity assay NF-kB transcription factor p65 and p50 DNA-binding activity was measured in 5 mg of nuclear extracts from stimulated BV173 and SUP-B15 cells by using a commercially available enzyme-linked immunosorbent assay kit (p50/p65 Transcription Factor Assay Kit; ab133128, Abcam, Cambridge, MA) according to the manufacturer’s instructions. Expression constructs and retroviral transductions The RAG-reporter construct was used as described previously.25 The retroviral RAG-reporter and IkBaSR (IkBa S32A/S36A) constructs were co-transfected with the pCL-ECO plasmid in Phoenix-A cells. For retroviral transduction, cells and retroviral supernatants were spun for 90 minutes at 2000g onto retronectin-coated 24-well plates (5 × 105 cells per well) according to the manufacturer’s instructions (Takara Bio Inc., Otsu, Shiga, Japan). Primary mouse bone marrow pre-B-cell cultures 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 were performed in agreement with national and institutional guidelines. Primary mouse pre-B-cell cultures were performed as described previously20; details are available in the supplemental Data. Patient samples Bone marrow aspirates and peripheral blood mononuclear cells from B-ALL patients containing >90% blasts were obtained and prepared according to ethical standards of our institutional medical ethical committee, as well as in agreement with the Helsinki Declaration of 1975, as revised in 1983. Cells were cultured for 48 hours in supplemented Iscove’s modified Dulbecco’s medium with 20% fetal calf serum, in the presence of 2.5 mM NF-kB kinase b (IKKb) inhibitor (IKKbi) and 2.5 mM AKT inhibitor (AKTi), or vehicle (dimethylsulfoxide; untreated). Patient cytogenetic characteristics are listed in SUPPL Table 2.
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