PMC

MDC1 is required to amplify the ATM response to hypoxia. (A) U20S cells were exposed to hypoxia for 6 h and then stained for MDC1 and RPA32. (B) YZ3 (ATM^+/+) and pEBS (ATM^−/−) cells were exposed to hypoxia (Hyp) for 16 h and then stained for MDC1, γ-H2AX, and DAPI. Con, control. (C) Cells were exposed to either 6 h of hypoxia or 6 Gy. Irradiated cells were fixed 4 h after treatment. Cells were stained for MDC1 and γ-H2AX, and nuclei were visualized using DAPI. (D) MDC1^−/− and MDC1^+/+ MEFs were exposed to hypoxia, and Western blotting was carried out for the proteins indicated.

The MRN complex is not required for the ATM response to hypoxia. LCLs of the genotypes shown were used for these experiments. (A) Cells were treated with NCS (5 ng/ml) for 10 min before harvesting. (B) A longer time course was then carried out with NCS (5 ng/ml). (C and D) Cells were then exposed to 0.02% O₂ for the times indicated, and Western blotting was carried out for the proteins/modifications shown. (E) RKO cells were exposed to hypoxia for the times indicated in the presence/absence of PARP inhibitor (PARPi), 4-amino-1,8-naphthalimide (10 μM). Western blotting was carried out for the proteins indicated. WT, wild type.

Mitochondrial signaling is not required for hypoxia-induced activation of ATM. (A) GM0536 (ATM^+/+) cells were exposed to 0.02% or 2% O₂ for 8 h in the presence of mitochondrial inhibitors (1 μM myxothiazol [Myx] or 10 μM antimycin A[AntA]). Western blotting was then carried out for ATM-S1981, HIF-1α, and p53-S15. Con, control. (B) A549 and A549 [rho⁰] cells were exposed to hypoxia (Hyp) for 16 h or NCS for 4 h. The levels of Kap1-S824 were then determined as a marker of ATM activity. wt, wild type. N, normoxic.

53BP1 and BRCA1 do not form nuclear foci in response to hypoxia. (A) U2OS cells were treated with hypoxia (6 h) or NCS (5 ng/ml 6 h) and stained for 53BP1 and γ-H2AX. (B) YZ3^(ATM+/+) and pEBS^(ATM−/−) cells were exposed to hypoxia for the times indicated, and Western blotting was carried out for p53BP1, 53BP1, and GAPDH. (C) RKO cells were exposed to hypoxia (Hyp) as shown, and Western blotting was carried out for the proteins and modifications indicated. (D) U2OS cells were exposed to hypoxia for 16 h followed by periods of reoxygenation; shown is 1 h of reoxygenation (Reox). Cells were then stained for 53BP1 and DAPI. U2OS cells were treated as described for panel A and stained for BRCA1 (E) and mono- and polyubquitinated substrates using the FK2 antibody (F). Con, control.

Hypoxia-induced ATM-S1981 is a result of an autophosphorylation event. (A) MO59K (DNA-PKcs^+/+), MO59J (DNA-PKcs^−/−, and MJ-L24 (complemented DNA-PKcs^+/+) cell lines were exposed to hypoxia, and Western blotting was carried out for ATM-S1981 and ATM. (B) GM0536 (ATM^+/+) cells were exposed to hypoxia (Hyp) for 7 h in the presence or absence of DNA-PK inhibitor II (10 μM) as indicated. The levels of total ATM and phosphorylated ATM are shown. (C) GM0536 (ATM^+/+), GM1526 (ATM^−/−), and DKOO64 (ATR/Seckel) were exposed to hypoxia for 6 h, and then Western blotting was carried out for total and phosphorylated ATM. (D) HCT116 or HCT116^ATR−/+ cells were exposed to 0.02% O₂ for 16 h. Western blotting was carried out for total and phosphorylated ATM. (E) GM0536 (ATM^+/+) cells were exposed to 0.02% O₂ for 6 h, plus and minus the ATM inhibitor (ATMi) (10 μM). The results of three identical experiments are also shown graphically. Con, control.

Hypoxia-induced γ-H2AX is dependent on ATR in S-phase cells. (A) pEBS (ATM^−/−) and YZ3 (ATM^+/+) cells were exposed to hypoxia for the time periods indicated. The levels of γ-H2AX are shown. (B) In an experiment parallel to the one shown in panel A, cells were also immunostained for γ-H2AX. (C) FO2/98/hTERT (Seckel) cells and a matched control, IBR hTERT, were treated as shown, and the induction of γ-H2AX was determined. (D) RKO cells were exposed to the oxygen tensions shown for 8 h. Western blotting was carried out for γ-H2AX and β actin. (E) U2OS cells were exposed to either 0.2% or 0.02% O₂ for 16 h. γ-H2AX-positive cells were then quantified with a fluorescence-activated cell sorter. (F) U2OS cells were exposed to hypoxia for 6 h or NCS for 6 h. Cells were then costained for γ-H2AX and CENP-F. (G) U2OS cells were exposed to hypoxia for 6 h and then stained for RPA32 and γ-H2AX. DAPI was used as a nuclear stain.

Hypoxia-activated ATM is nuclear but does not form foci. (A) GM0536 cells were treated with NCS (5 ng/ml) for 20 min or hypoxia (Hyp) for 8 h. Cell fractionation was then carried out to isolate cytoplasmic (Cyt) or nuclear (N) preparations. MRE11 was used as a nuclear marker, and tubulin was used for the cytoplasm. (B) Cell fractionation was carried out as described previously (). HeLa cells were treated for 1 h with 5 ng/ml NCS or hypoxia (18 h). The levels of ATM and HIF-1α are shown for fractions I and III. (C and D) ATM does not form large nuclear foci in response to hypoxia. YZ3 (ATM^+/+) and pEBS (ATM^−/−) cells were exposed to either hypoxia (18 h) or NCS (2 h, 5 ng/ml). Cells were then stained for total ATM protein (C) or ATM-S1981 (D).

ATM is phosphorylated and active during both hypoxia and reoxygenation. GM0536 cells were exposed to the levels of hypoxia shown, to hypoxia-mimetic CoCl₂ for 6 h, or, as a positive control, to the DNA-damaging agent NCS (5 ng/ml) for 10 min. Shown is a Western blot for the proteins or protein modifications indicated. (A) GAPDH is shown as a loading control. (B) HCT116^wt and HCT116^{HIF-1α−/−} cells were exposed to hypoxia (Hyp) for 16 h or NCS for 2 h. Western blotting was carried out for the proteins indicated. (C) The GM0536 cell line was exposed to hypoxia as shown and then reoxygenated for the time periods indicated. The levels of phosphorylated and total ATM are shown. (D) LCLs from either a healthy control (GM0536) or an AT patient (GM1526) were exposed to hypoxia for the times indicated, and Western blotting was carried out for Kap1-S824, DNA-PKcs-T2609, and GAPDH as a loading control. (E) GM0536 cells were exposed to hypoxia for 8 h in the presence of the ATM inhibitor (ATMi) (10 μM). Cells were also treated with NCS for 10 min as a positive control. The levels of phosphorylated Kap1 (residue serine 824) are shown. wt, wild type; N, normoxic.