4 DNA damage response regulates RAG1/2 expression through ATM-FOXO1 | 89 Proximity ligation assay Protein colocalizations/interactions in cells were determined by the Duolink® proximity ligation assay as per manufacturer’s directions (Sigma-Aldrich). BV173 cells exposed to different treatments were cytocentrifuged onto glass slides, fixed for 30 minutes in 4% paraformaldehyde, permeabilized in 0.25% Triton-X, washed and blocked in Duolink® blocking solution prior to overnight incubation with primary antibody combinations. The following combinations were applied: mouse-anti-phospho-ATM (Ser1981) (10H11.E12, EMD MilliPore) + rabbit-anti-FOXO1 (C29H4, Cell Signaling Technology), goat-anti-ATM (A300-136A, PLA-grade, Bethyl Laboratories Inc. Montgomery, TX) + rabbit-anti-FOXO1, goat-anti-ATM + rabbit-anti phospho-p53 (Ser15) (9284, Cell Signaling Technology). After washing, the slides were incubated with combinations of plus- and minus-PLA-probes (anti-mouse + anti-rabbit or anti-goat + anti-rabbit), followed by ligation and rolling circle amplification using the Duolink® in situ Red kit (Sigma-Aldrich). Nuclei were counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and slides were mounted with VECTASHIELD antifading mounting medium (Vectorlabs, Burlingame, CA). Protein interactions were quantified by assessing the number of PLA-foci in at least 20 cells for each experimental condition. Imaging was performed using a Leica DM5000B fluorescence microscope equipped with a Leica FDC500 camera and a 20x objective (Leica Microsystems BV, Son, the Netherlands). Statistics The GraphPad Prism software package (GraphPad Software, La Jolla, CA) was used for statistical testing. Results DNA breaks regulate RAG1 expression in pre-B cells To assess the role of DNA damage in the regulation of RAG1 expression, BCR-ABL-positive human pre-B cells and v-Abl-transformed mouse pre-B cells were treated overnight with the specific Abl kinase-inhibitor STI571 (imatinib methanesulfonate) to induce RAG expression and Igl recombination and were subsequently exposed to the radiomimetic drug neocarzinostatin (NCS) for 2 hours to induce DNA damage. NCS rapidly induces chromosome breaks in quiescent cells, not requiring S-phase for its DNA-damaging effect28. Such short exposure of pre-B cells to this DNA damaging agent did not result in detectable cell death at the time of sample collection, judging by the percent of apoptotic (propidium-iodide positive) cells as detected by flow cytometry (SUPPL Figure 1A), but generated substantial DNA damage as the levels of phospho-p53 (Ser15) and phospho-ATM (Ser1981) were strongly induced (Figure 1A). In addition, p21 mRNA expression was increased by 13.5
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