Abnormal ATM–p53 axis function in Mule-deficient B cells. (A) Purified splenic control and BMKO B cells were treated with γ-irradiation (5Gy) for 2 h (left), or 10 µM etoposide for 3 or 6 h (right), followed by immunoblotting to detect phospho-p53-Ser18 (S18-p53). (B) Purified splenic littermate control and BMKO B cells were γ-irradiated (5 Gy) followed by immunoblotting to detect phospho-ATM-Ser1987 (S1987-ATM) at the indicated times after irradiation. (C) Purified splenic control and BMKO B cells were treated as in B. Total Brca1 was detected by immunoblotting. (D) Purified splenic control and BMKO B cells were treated with 10 µM etoposide (Etop) for 6 h and degradation of Mcl-1 was monitored by immunoblotting. M/A is the ratio of Mcl-1 protein to actin protein as determined by densitometry (the ratio in unstimulated controls was defined as 1). (E) Purified splenic littermate control and BMKO B cells were treated with 10 µM etoposide for 2, 4, or 6 h followed by detection of cleaved (activated) caspase-3 by flow cytometry. Numbers are the percentage of B cells positive for cleaved caspase-3. Results are representative of a single experiment involving three mice per genotype. (F) Purified splenic littermate control and BMKO B cells were treated with the indicated doses of etoposide or dexamethasone (Dex) for 20–24 h and viability was determined by flow cytometric analysis of Annexin V versus propidium iodide or 7AAD (7-amino-actinomycin D) expression. Left, representative flow cytometry plot. Numbers are percentages of viable cells. Right, quantitation of viability. Results are the combined mean ± SD of three to five independent experiments each involving one to three mice per genotype (**, P < 0.005; ns, not significant). For A–D, results are representative of two to three independent experiments.