Increased senescence in macrophages from Nbs1^∆B/∆B mice. (A) Shorter average telomere length in Nbs1^∆B/∆B BMDMs. DNA from BMDMs was extracted and relative telomere length was determined by qPCR. (B) Metaphase spreads were performed as indicated in the “Materials and methods” section. Quantification of metaphase aberrations in 100 cells per mouse is shown (n = 3). (C) The expression of p21^waf-1 was determined by qPCR from BMDMs stimulated with proinflammatory (IFN-γ) or proliferative (M-CSF) stimuli for the indicated times. (D) BMDMs were stimulated with M-CSF for 30 hours, and ROS levels were determined by flow cytometry using dichlorofluorescin diacetate (DCF-DA) as an indicator. (E) BMDM cells were activated with IFN- γ for 24 hours, and then the expression of MHC class II was determined by flow cytometry. (F) The macrophages from Nbs1^∆B/∆B mice express higher levels of proinflammatory cytokines. BMDMs were incubated for 6 hours with the indicated stimuli, and the expression of Il-6, Ifn-β, and Tnf-α was determined by qPCR. Each point was performed in triplicate and the results are shown as mean ± SD. All assays are representative of at least 4 independent experiments showing similar results. *P < .05, **P < .01, and ***P < .001 in relation to the controls when all the independent experiments had been compared. MFI, mean fluorescent intensity.