Figure 5
Figure 5. Allelic loss of Beclin-1 rescues mitochondrial abnormalities in ATM-null thymocytes. (A) Allelic loss of Beclin-1 partially reverses the increase in mitochondrial mass observed in ATM−/− mice. Freshly isolated thymic cells were stained with 5nM NAO dye. A representative histogram of NAO fluorescence intensity is illustrated. Graphs illustrate the average mean intensity of NAO for each genotype (n ≥ 3/genotype). (B) Beclin-1 heterozygosity reverses the abnormal increase in mtDNA observed in ATM−/− thymocytes. The ratio of mitochondrial/nuclear DNA was quantified in viable thymic cells of mice with desired genotypes by real-time PCR using primers for mitochondrial ND2 and nuclear 18S rRNA genes. Mean values are shown (n ≥ 3/genotype). (C-D) Beclin-1 heterozygosity reverses defects in complex I activity and mROS levels in ATM−/− thymocytes. Complex I activity (C) and mROS levels (D) were measured as described in Figure 1. For complex I activity, the relative activities of desired genotypes were graphed. For mROS, a representative histogram of MitoSOX fluorescence intensity per genotype is illustrated. Graph shows the averaged mean intensity for each genotype (n ≥ 3/group). P values were obtained by implementing the Student t test analysis in experiments shown in panels A-E (*P ≤ .04, **P ≤ .01, ***P ≤ .001). (E) Allelic loss of Beclin-1 rescues the increase in cellular respiration observed in the ATM−/− mice. The rate of oxygen consumption was measured in viable thymic cells of desired genotypes (n = 3/group). Shown is the OCR of thymic cells as function of time in minutes. Equal numbers of viable cells were used per sample for these experiments. (F-G) Allelic loss of Beclin-1 reduces mitochondrial ultra-structure abnormalities in ATM-null thymocytes. (F) Whole thymii of 5- to 8-week-old mice of the indicated genotypes were analyzed by transmission electron microscopy, and representative photomicrographs are shown. Mitochondria with disrupted cristae structure and with swollen appearance were considered abnormal. At least 15 images (magnification, ×10 000) were scored per sample. Red arrowheads illustrate abnormal mitochondria having disorganized cristae structure seen in ATM−/− thymic cells. The black arrow denotes an autophagic vacuole. (G) Percentage of abnormal mitochondria quantified in the whole thymus of mice shown in panel F. Each dot, triangle, or square represents a single mouse. Statistical analyses were performed using the Student t test (*P ≤ .03).

Allelic loss of Beclin-1 rescues mitochondrial abnormalities in ATM-null thymocytes. (A) Allelic loss of Beclin-1 partially reverses the increase in mitochondrial mass observed in ATM−/− mice. Freshly isolated thymic cells were stained with 5nM NAO dye. A representative histogram of NAO fluorescence intensity is illustrated. Graphs illustrate the average mean intensity of NAO for each genotype (n ≥ 3/genotype). (B) Beclin-1 heterozygosity reverses the abnormal increase in mtDNA observed in ATM−/− thymocytes. The ratio of mitochondrial/nuclear DNA was quantified in viable thymic cells of mice with desired genotypes by real-time PCR using primers for mitochondrial ND2 and nuclear 18S rRNA genes. Mean values are shown (n ≥ 3/genotype). (C-D) Beclin-1 heterozygosity reverses defects in complex I activity and mROS levels in ATM−/− thymocytes. Complex I activity (C) and mROS levels (D) were measured as described in Figure 1. For complex I activity, the relative activities of desired genotypes were graphed. For mROS, a representative histogram of MitoSOX fluorescence intensity per genotype is illustrated. Graph shows the averaged mean intensity for each genotype (n ≥ 3/group). P values were obtained by implementing the Student t test analysis in experiments shown in panels A-E (*P ≤ .04, **P ≤ .01, ***P ≤ .001). (E) Allelic loss of Beclin-1 rescues the increase in cellular respiration observed in the ATM−/− mice. The rate of oxygen consumption was measured in viable thymic cells of desired genotypes (n = 3/group). Shown is the OCR of thymic cells as function of time in minutes. Equal numbers of viable cells were used per sample for these experiments. (F-G) Allelic loss of Beclin-1 reduces mitochondrial ultra-structure abnormalities in ATM-null thymocytes. (F) Whole thymii of 5- to 8-week-old mice of the indicated genotypes were analyzed by transmission electron microscopy, and representative photomicrographs are shown. Mitochondria with disrupted cristae structure and with swollen appearance were considered abnormal. At least 15 images (magnification, ×10 000) were scored per sample. Red arrowheads illustrate abnormal mitochondria having disorganized cristae structure seen in ATM−/− thymic cells. The black arrow denotes an autophagic vacuole. (G) Percentage of abnormal mitochondria quantified in the whole thymus of mice shown in panel F. Each dot, triangle, or square represents a single mouse. Statistical analyses were performed using the Student t test (*P ≤ .03).

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