American Society of Hematology

Figure 6

$Figure 6. ATM localizes to mitochondria and becomes activated by mitochondrial dysfunction without evidence of DNA damage. (A) ATM protein localizes to mitochondria. Normal human fibroblasts were fractionated into nuclear (N), cytoplasmic (C), and mitochondrial (M) fractions and the basal levels of total and phospho-ATM (S1981) were analyzed by immunoblotting. Topoisomerase I (Topo), lactate dehydrogenase (LDH), and COX-IV antibodies were used as N, C, and M markers, respectively. We also analyzed 10 μg of total (T) protein. (B) ATM is activated in response to dysfunctional mitochondria without evidence of DNA damage induction. Viable thymic cells isolated from 6- to 8-week-old wild-type (WT) or ATM-null mice were either treated with DMSO or 50μM of the mitochondrial uncoupler CCCP for 3 hours followed by total protein isolation and analysis by immunoblotting for various ATM substrates, including P-KAP1, P-SMC1, total p53, P-p53, and P-ATM itself. As a positive control for ATM activity in vivo, total protein lysates from irradiated (6 Gy) or nonirradiated control mice were included in the assay. LC3-II also was analyzed to confirm increased basal autophagy in ATM-null cells and induction of this process by CCCP treatment. (C-D) Transient inhibition of ATM results in mitochondrial mass abnormalities. (C) Wild-type thymocytes were treated with DMSO alone or with 10μM CP (ATM kinase inhibitor) for 3 or 5 hours before staining with MitoTracker Green FM (MTG) for 15 minutes. Flow cytometry histograms of 10 000 viable cells per sample are shown. (D) Control or A-T human fibroblasts were treated with either DMSO or 10μM CP for 3 hours before staining with MTG dye and analysis by flow cytometry. Shown is the mean of fluorescence intensity (MFI) in each sample.$

ATM localizes to mitochondria and becomes activated by mitochondrial dysfunction without evidence of DNA damage. (A) ATM protein localizes to mitochondria. Normal human fibroblasts were fractionated into nuclear (N), cytoplasmic (C), and mitochondrial (M) fractions and the basal levels of total and phospho-ATM (S1981) were analyzed by immunoblotting. Topoisomerase I (Topo), lactate dehydrogenase (LDH), and COX-IV antibodies were used as N, C, and M markers, respectively. We also analyzed 10 μg of total (T) protein. (B) ATM is activated in response to dysfunctional mitochondria without evidence of DNA damage induction. Viable thymic cells isolated from 6- to 8-week-old wild-type (WT) or ATM-null mice were either treated with DMSO or 50μM of the mitochondrial uncoupler CCCP for 3 hours followed by total protein isolation and analysis by immunoblotting for various ATM substrates, including P-KAP1, P-SMC1, total p53, P-p53, and P-ATM itself. As a positive control for ATM activity in vivo, total protein lysates from irradiated (6 Gy) or nonirradiated control mice were included in the assay. LC3-II also was analyzed to confirm increased basal autophagy in ATM-null cells and induction of this process by CCCP treatment. (C-D) Transient inhibition of ATM results in mitochondrial mass abnormalities. (C) Wild-type thymocytes were treated with DMSO alone or with 10μM CP (ATM kinase inhibitor) for 3 or 5 hours before staining with MitoTracker Green FM (MTG) for 15 minutes. Flow cytometry histograms of 10 000 viable cells per sample are shown. (D) Control or A-T human fibroblasts were treated with either DMSO or 10μM CP for 3 hours before staining with MTG dye and analysis by flow cytometry. Shown is the mean of fluorescence intensity (MFI) in each sample.

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