1. Volume 16, Issue 5, Pages 715-724 (December 2004)
A Pathway of Double-Strand Break Rejoining Dependent upon ATM, Artemis, and Proteins Locating to γ-H2AX Foci 
Enriqueta Riballo, Martin Kühne, Nicole Rief, Aidan Doherty, Graeme C.M. Smith, Marı́a-José Recio, Caroline Reis, Kirsten Dahm, Andreas Fricke, Andrea Krempler, Antony R. Parker, Stephen P. Jackson, Andrew Gennery, Penny A. Jeggo, Markus Löbrich 
Molecular Cell 
Volume 16, Issue 5, Pages (December 2004)
DOI: /j.molcel

2. Figure 1
ATM- and Artemis-Deficient Cells Exhibit an Identical DSB Repair Defect
(A) PFGE analysis in control fibroblasts (MRC-5) and lines defective in ATM (AT1BR) or Artemis (CJ179, CJ179hTert). Confluent nondividing cells were irradiated with 80 Gy X-rays and incubated for 24 hr (left panel) or 21 days (right panel). Similar results were obtained after 40 Gy γ-rays (data not shown).
(B) γ-H2AX analysis after 2 Gy X-irradiation. HSF2 is another control fibroblast line. Without irradiation, all cell lines had less than 0.5 foci/cell. Similar results were obtained following 2 Gy γ-rays.
(C) Artemis-defective cell lines (CJ179, DB333, F01-240, and F02-385) show a DSB repair phenotype similar to an A-T line (AT7BI) and distinct from a control (1BR3). γ-H2AX analysis was performed after 1.3 Gy γ-irradiation. The mean and SEM of three experiments is shown. Initial foci were assessed at 15 min post IR.
(D) γ-H2AX analysis after 2 Gy γ-irradiation in CJ179 cells following transfection with vector cDNA, c-Myc-Artemis cDNA, or D37N c-Myc-Artemis cDNA. Transfection resulted in a small increase in γ-H2AX foci in unirradiated cells likely due to the introduction of DNA ends. The lack of full correction is most likely due to inefficient expression of c-Myc-Artemis cDNA in the primary cells. Transfection of c-Myc-Artemis cDNA in 1BR3 cells did not affect γ-H2AX numbers compared to cells transfected with c-Myc-vector (data not shown). With the exception of the analysis of the D37N mutant (n = 3), error bars represent the SEM from six experiments.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Epistasis Analysis Shows that ATM, Artemis, and DNA-PK Function in the Same Pathway
(A) DSB repair after 2 Gy X-irradiation in the presence or absence of KU (ATMi). Initial foci were assessed at 30 min post IR. Error bars represent the SEM from two to three experiments. If not visible, they lie within the symbols.
(B) γ-H2AX analysis in wild-type, ATM−/−, DNA-PKcs−/−, and LIG4−/− MEFs after 2 Gy X-irradiation demonstrating a repair defect in ATM−/− MEFs. Initial foci were assessed at 15 min post IR. Without irradiation, the number of foci/cell was below 0.5 for all cell lines. Since MEFs cannot be held in confluency for prolonged periods, foci analysis was undertaken maximally at 3 days post irradiation.
(C) DSB repair after 2 Gy X-irradiation in the presence or absence of the ATM inhibitor KU (ATMi). Wild-type MEFs treated with KU show a repair defect similar to ATM−/− MEFs (compare with [B]). LIG4−/− MEFs exhibit the same level of unrepaired DSBs with and without KU treatment. The counting of these foci was computer assisted (see Supplemental Data for further details).
(D) DSB repair after 2 Gy X-irradiation in the presence or absence of the DNA-PKcs inhibitor NU7026 (PKi). Wild-type MEFs treated with NU7026 show a repair defect similar to DNA-PKcs−/− MEFs, and NU7026 treatment of DNA-PKcs−/− MEFs has no effect.
(E) DSB repair after 2 Gy X-irradiation in primary human fibroblasts in the presence or absence of NU7026. Control cells treated with NU7026 show a repair defect greater than that of Artemis cells. Artemis cells treated with NU7026 exhibit the same level of unrepaired DSBs as NU7026-treated control cells. Initial foci were assessed at 30 min post IR. Error bars represent the SEM from two to three experiments. If not visible, they lie within the symbols.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Nbs1, Mre11, 53BP1, and H2AX Are Required for ATM-Dependent NHEJ
(A) γ-H2AX foci analysis at 1 day post 2 Gy X-rays shows a repair defect in ATLD (ATLD2) and NBS (CZD82CH) cells. Addition of KU (ATMi) and NU7026 (PKi) to ATLD2 and CZD82CH does not cause additivity in foci numbers. Without irradiation, the numbers of foci per cell were below 0.5 for all lines.
(B) γ-H2AX analysis at 1 day post 2 Gy in wild-type, Chk2−/−, and 53BP1−/− MEFs. Addition of KU (ATMi) and NU7026 (PKi) to 53BP1−/− MEFs does not cause additivity in foci numbers. Chk2−/− MEFs respond like wild-type MEFs.
(C) PFGE analysis in wild-type, ATM−/−, H2AX−/−, DNA-PKcs−/−, and LIG4−/− MEFs after 80 Gy X-irradiation. H2AX−/− MEFs show a repair defect similar to that of ATM−/− MEFs. The panel displays the fraction of activity released (FAR) versus repair time (in contrast to Figure 1A, which shows percentage of unrepaired DSBs versus repair time) to allow a comparison with Figure 4C (see Supplemental Data). Error bars represent the SEM from two to four experiments.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
The Fraction of ATM-Dependent DSB Repair Depends on the Nature of the Break
(A) γ-H2AX analysis in control fibroblasts (MRC-5, HSF2) and lines defective in ATM (AT1BR) or Artemis (CJ179) after exposure to 2 Gy α particles. The fraction of unrepaired DSBs (DSBs remaining/DSBs induced) in AT1BR and CJ179 is 2-fold higher than after X-irradiation (compare with Figure 1B).
(B) γ-H2AX analysis in control fibroblasts (48BR) and lines defective in LIG4 (180BR), ATM (AT7BI), or Artemis (F01-240) after exposure to 20 μM etoposide. The concentration of etoposide used induces the same number of γ-H2AX foci as 1.3 Gy γ-rays. No repair defect is observed in ATM- and Artemis-deficient cells (compare with Figure 1C).
(C) PFGE analysis in wild-type, ATM−/−, H2AX−/−, DNA-PKcs−/−, and LIG4−/− MEFs after exposure to 500 μM etoposide. In agreement with (B), no DSB repair defect can be observed in ATM−/− MEFs. H2AX−/− MEFs also fail to show a repair defect. The relative requirements for DNA-PKcs and LIG4 in DSB repair after etoposide treatment are similar to those after IR (compare with Figure 3C). Error bars represent the SEM from two to three experiments.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
ATM-Dependent DSB Rejoining Substantially Contributes to Survival and Affects the Repair of Potentially Lethal Damage
(A) A-T (AT1BR, AT5BI) and Artemis (CJ179, DB333, F02-385) lines show similar radiosensitivity.
(B) H2AX−/− and 53BP1−/− MEFs show similar sensitivity to ATM−/− MEFs. LIG4−/− MEFs, which show a dramatic defect in DSB repair, are more radiosensitive.
(C) Control fibroblasts (1BR3) and lines defective in Artemis (CJ179) were either plated immediately (IP) or 1 day after irradiation (DP). CJ179 does not show significant recovery in delayed plating experiments. Error bars represent the SEM from three experiments.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
ATM Is Required for Radiation-Induced Artemis Hyperphosphorylation, and Artemis Coassociates with 53BP1
(A) KU (ATMi) inhibits IR-induced Artemis hyperphosphorylation. MRC5Vi cells (transformed MRC-5) were stably transfected with c-Myc-Artemis cDNA. Where indicated, KU or NU7026 (DNA-PKi) was added 1 hr prior to irradiation. Samples were analyzed 1 hr post irradiation with 2 Gy γ-rays (lower panel). The upper panel shows control cells (no drug treatment) irradiated with 10 Gy and treated with phosphatase.
(B) IR-induced Artemis hyperphosphorylation is not observed in A-T cells. AT5BIVa cells (transformed AT5BI) were transiently transfected with c-Myc-Artemis cDNA. 48 hr post transfection, cells were irradiated with 10 Gy and analyzed 1 hr later.
(C) Analysis of IR-induced Artemis hyperphosporylation in wild-type, ATM−/−, and DNA-PKcs−/− MEFs. MEFs were transiently transfected with c-Myc-Artemis cDNA. 48 hr post transfection, cells were irradiated with 10 Gy and analyzed 1 hr later. Note that (B) and (C) used a Western blotting system distinct from (A) to maximize the separation between the forms of Artemis.
(D) Artemis coassociates with 53BP1. MRC5Vi cells were stably transfected with c-Myc-Artemis (Artemis) or c-Myc (Vector) cDNA. WCE (without irradiation) was immunoprecipitated using α-53BP1 or α-c-Myc and subjected to Western blotting using α-c-Myc or α-53BP1. Interaction was observed only in cells expressing c-Myc-Artemis cDNA. The c-Myc-Artemis band observed migrated at the expected ∼100 kDa. Immunoprecipitation was carried out in the presence of ethidium bromide.
Molecular Cell  , DOI: ( /j.molcel )