Rescue of Rad50^S/S survival by Rad50-null, Atm-null, Mre11^ATLD1, and Nbs1^ΔB alleles. (A) Kaplan-Meier survival curves of Rad50^S/S and Rad50^Δ/S mice. (B) Kaplan-Meier survival curves of Rad50^S/S, Atm^-/-, Rad50^S/S Atm^+/-, and Rad50^S/S Atm^-/- mice. (C) Kaplan-Meier survival curves of Rad50^S/S, Rad50^S/S Mre11^+/ATLD1, Rad50^S/S Mre11^ATLD/ATLD1, Rad50^S/S Nbs1^+/ΔB, and Rad50^S/S Nbs1^ΔB/ΔB mice. (A–C) In each table below the survival curves, for each cohort, the number of mice that were found dead without overt signs of anemia and malignancy is equal to the number of mice minus the number of alive mice, the number of dead mice with anemia, and the number of dead mice with lymphoma. (D) Hematoxylin and eosin staining (upper pictures) or cleaved Caspase-3 detected by immunohistochemistry (lower pictures) in bone marrow sections from 4-wk-old Rad50^S/S, Rad50^S/S Atm^-/-, Rad50^S/S Mre11^+/ATLD1, and Rad50^S/S Mre11^ATLD/ATLD1 mice. Magnification, 100×. Bar, 100 μM.

Rescue of Rad50^S/S hematopoietic attrition by Atm-null, Mre11^ATLD1, and Nbs1^ΔB alleles, and by Chk2^-/-. Flow cytometric analysis of hematopoietic tissues from 4–9-wk-old mice. The numbers of double-negative T cells in the thymus (DN T cells; A), and macrophages (B) and pro-B cells in the bone marrow (C), normalized to wild-type data, are depicted for the indicated genotypes.

Rescue of Rad50^S/S survival by Chk2-null allele and the Eμ-Bcl2 transgene. (A) Kaplan-Meier survival curves of Rad50^S/S and Rad50^S/S Eμ-Bcl2 mice. (B) Kaplan-Meier survival curves of Rad50^S/S, Rad50^S/S Chk2^+/-, Rad50^S/S Chk2^-/-, and Rad50^S/S Smc1^2SA/2SA mice. Each table below the survival curves is as in Figure 1.

Constitutive ATM activity in Rad50^S/S is rescued by ATM deficiency and Mre11^ATLD1. (A) Analysis of ATM Ser1981 phosphorylation. Extracts from wild-type (WT), Rad50^S/S, Rad50^S/S Mre11^ATLD1/ATLD1, and Atm^-/- p4 ear fibroblasts were prepared after mock treatment and 1 h after 10 Gy of IR. For the ATM Ser1981 phosphorylation Western blot (ATM-Ser1981-P), the lower reactive species represents ATM, as indicated by the ATM Western blot (ATM), and the upper band is nonspecific. SMC1 is included as a loading control. (B) Analysis of 53BP1 phosphorylation in primary ear fibroblasts. Ser25-phosphorylated 53BP1 was immunoprecipitated from untreated or irradiated (10 min after 4 Gy) primary ear fibroblast extracts, and detected with a 53BP1 antibody. A 53BP1 Western blot of the same extracts is shown as a loading control. (C) Analysis of SMC1 Ser 957 phosphorylation in the indicated mutants. Extracts from p4 ear fibroblasts were prepared after mock treatment and 1 h after 10 Gy of IR. The extracts were sequentially immunoblotted with SMC1-Ser957-P and SMC1 (loading control) antisera. (WT) Mock-treated wild-type lane underloaded for SMC1. As untreated Mre11^+/ATLD1 cells were equivalent to wild type for the levels of SMC1 phosphorylation (data not shown), compare mutants with that lane.

DNA damage signaling is enhanced in Rad50^S/S Atm^-/- cells compared with Atm^-/- cells. (A) SQ/TQ foci formation in wild-type (WT), Atm^-/-, Rad50^S/S, and Rad50^S/S Atm^-/- primary ear fibroblasts was assessed by immunofluorescence. Primary ear fibroblasts were harvested 30 min after mock treatment (white bars) or 4 Gy of IR (gray bars). Two-hundred-fifty cells were counted for presence or absence of SQ/TQ foci. A cell is considered positive when it shows ≥10 foci. Data are from three experiments. (B) SQ/TQ foci from mock-treated or 4 Gy-irradiated primary ear fibroblasts. (C) Primary ear fibroblasts were pretreated for 1 h with caffeine (20 mM) or vehicle and harvested 30 min after treatment with 4 Gy IR as indicated. For each treatment, 250 cells were counted for presence or absence of SQ/TQ foci. Data are from two experiments run in duplicate. (D) Extracts from primary MEF cultures prepared after mock treatment (-); 1 h after 1 μM CPT, 2 mM HU, or 20 J/m² UV; or 10 min after 5 Gy of IR treatment were sequentially immunoblotted with γ-H2AX and Actin (loading control) antisera. “Short” and “Long” represent short and long exposure, respectively.

Rad50^S rescues the growth defects and IR-sensitivity of Atm^-/- cells. (A) Cumulative growth curve. Passage 2 MEFs from two cultures were seeded in triplicate onto 6-well plates, counted every 3 d, and replated for four passages. Genotypes as indicated. (B,C) IR sensitivity determined by colony forming assay. (B) The surviving fractions of wild-type (WT), Atm^-/-, Rad50^S/S, and Rad50^S/S Atm^-/- SV40-transformed MEFs after various doses of IR are shown. Note that the wild-type and Rad50^S/S curves overlap. Graph contains data from three independent experiments in which cells were plated in triplicate at each dose. Similar results were obtained with SV40-transformed ear fibroblasts. (C) The surviving fractions of wild-type (WT), Mre11^ATLD/ATLD1, Rad50^S/S, Rad50^S/S Mre11^+/ATLD1, and Rad50^S/S Mre11^ATLD/ATLD1 SV40-transformed MEFs after various doses of IR are shown.