Allelic loss of Beclin-1 does not affect DNA damage responses. (A) Both ATM^−/− and ATM^−/−Beclin-1^+/− mice display radiosensitivity. Survival of control, ATM^−/−, and ATM^−/−Beclin-1^+/− mice exposed to 8-Gy total body irradiation is shown (P = .85 between ATM^−/− and ATM^−/−Beclin-1^+/− mice, log-rank (Mantel-Cox) test. (B-D) The DDR pathway is equally defective in ATM^−/− and ATM^−/−Beclin-1^+/− mice. (B) Immunoblotting of total protein, phosphorylation status, or both of various ATM substrates, including P-KAP1, P-SMC1, total p53, P-p53, and P-ATM itself in thymocytes from mice sacrificed 2 hours after being exposed to 6-Gy irradiation. Actin was used as loading control. (C) Thymic tissues were isolated from nonirradiated or whole body–irradiated animals (6 Gy/1 hour) and embedded in paraffin followed by immunofluorescence staining against the genomic instability marker γH2AX (green). 4,6-diamidino-2-phenylindole (blue) was used to visualize nuclei (magnification, 40×0.055). (D) The lack of rescue of the DDR abnormalities by Beclin-1 heterozygosity is not because of a delay of DDR signaling. Animals were irradiated with 6 Gy, and tissues were harvested 7 hours later followed by isolation of total proteins from thymocytes. The expression or phosphorylation status of proteins involved in DDR (P-KAP, total-p53, P-p53, and P-ATM) was analyzed. Both ATM^−/− and ATM^−/−Beclin-1^+/− thymocytes showed a mild increase in the levels of proteins involved in DDR (P-KAP, total-p53, and P-p53) compared with cells exposed for 2 hours (B), but no difference was observed between these 2 genotypes at this time point (D). Actin served as loading control.