1. Separation-of-function mutations in ATM dictate responses to DNA damage and oxidative stress.
Separation-of-function mutations in ATM dictate responses to DNA damage and oxidative stress. Human U2OS osteosarcoma cells were depleted for endogenous ATM using short hairpin RNA (shRNA) and induced to express various ATM alleles as indicated. (A) Cells were induced for ATM expression with doxycycline (10 ng/ml) and exposed to camptothecin (CPT) (10 μM) for 1 hour. ATM activity was examined using antibodies directed against phospho-Kap1 Ser824, and ATM levels were assessed with anti-ATM antibody. (B) U2OS cells were treated as in (A) except that expression was induced with doxycycline (1 μg/ml). ATM activity was assessed with antibodies directed against ATM, phospho-ATM Ser1981, Kap1, phospho-Kap1 Ser824, Chk2, and phospho-Chk2 Thr68 as indicated. (C) U2OS cells expressing shRNA against ATM (shATM) and either vector (CTRL) or various ATM alleles as in (B) were treated with 10 μM CPT for 1 hour, and the levels of phosphorylated Kap1 were determined, in comparison to total Kap1 protein, and normalized with the phosphorylated signal from WT ATM–expressing cells. NS, not significant. (D) U2OS cells were depleted of endogenous ATM and induced for recombinant ATM expression as in (B) but were exposed to IR (10 Gy) followed by 1-hour recovery. Phosphorylation was assessed as in (B). (E) U2OS cells were treated as in (B) but exposed to 100 μM H2O2 or arsenite for 1 hour in the presence of doxycycline (1 μg/ml). Phosphorylation was assessed as in (B). (F) U2OS cells expressing various ATM alleles as in (B) were treated with 100 μM H2O2 for 1 hour in serum-free medium, and the amount of phosphorylated Chk2 was quantitated in comparison to total Chk2 protein and normalized with the phosphorylated signal from WT ATM–expressing cells. Data are means ± SD of three independent experiments. *P < 0.05 and **P <
Ji-Hoon Lee et al., Sci. Signal. 2018;11:eaan5598
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