Mitochondrial dysfunction in ATM-null thymocytes. (A) Ultrastructural abnormalities in ATM-deficient thymic cells. Representative pictures taken by transmission electron microscopy in young mice from wild-type or ATM^−/− mice. ATM^−/− thymic tissue showed a number of mitochondria with disorganized structure and swollen appearance. Arrowheads denote mitochondria. Magnification, × 6000. Boxes show higher magnifications of the mitochondria. (B-C) Increased mitochondrial mass and mROS in ATM^−/− thymocytes. Freshly isolated thymic cells were stained with 200nM MitoTracker Green (MTG) probe (B) or 5μM superoxide indicator MitoSOX (C) and analyzed by flow cytometry. Representative histograms of MTG and MitoSOX fluorescence intensity per genotype are illustrated. Graph shows the averaged mean intensity for each genotype (n ≥ 3/group; Student t test: **P ≤ .001, ***P ≤ .0001). (D-E) ATM-deficient thymocytes display reduced activity of complex I of the ETC and diminished levels of cellular ATP. (D) Total protein was extracted from freshly isolated viable thymocytes of 5- to 8-week-old mice of the indicated genotypes. The activity of complex I of the ETC was analyzed using equal amount of protein per sample (Student t test: *P ≤ .02; n ≥ 3/genotype). (E) ATM^−/− thymocytes display reductions in cellular ATP levels. Total cellular ATP levels were measured in equal number of freshly isolated viable thymic cells isolated from 5- to 8-week-old mice of the indicated genotypes. Shown are the relative ATP levels for each cohort (Student t test: *P ≤ .02; n ≥ 3/genotype).