Reduced proliferation of macrophages from Nbs1^∆B/∆B mice. (A) The number of total cells from the BM at day 0 of differentiation (BM after it was flushed from bones) and after 6 days of differentiation (in media containing M-CSF) from the indicated genotypes. (B) Macrophages were starved of growth factor for 18 hours, incubated for 1 hour with the indicated stimuli (LPS or IFN-γ), washed, and incubated for 24 hours with M-CSF (10 ng/mL) in a hyperoxic (20% O₂) or hypoxic (1% O₂) environment, and proliferation was quantified by incorporation of tritium. (C) Macrophages were starved of growth factor for 18 hours and incubated for 24 hours with M-CSF. Cell cycle distribution was determined by staining fixed cells with PI and assessment by flow cytometry. (D) Impaired NBS1 function does not induce cell death in macrophages treated with M-CSF or IFN-γ. Cells were treated as in (B), and viability was determined by using PI staining and flow cytometry. (E) The levels of Bcl2 mRNA expression were determined by qPCR in BMDMs treated with M-CSF or IFN-γ for the indicated times. Each experiment 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 < .01 and ***P < .001 in relation to the controls when all the independent experiments had been compared.