Fig 7.

Ku70 S155A substitution is required and sufficient to confer increased survival following IR. (A) Clonogenic assay of Ku70^−/− MEFs expressing Ku70 with alanine substitutions at positions 155 to 160 (155-160A) and S155A/D156A and Ku70^−/− expressing wild-type Ku70 (WT). Survival is expressed as the number of colonies present at each IR dose relative to the unirradiated control, averaged for three experiments, with error bars representing the SD (∗∗, P < 0.01; ∗, P < 0.05). (B) Clonogenic assay comparing the survival of MEFs expressing wild-type Ku70 (WT) and Ku70 D156A and S155A substitutions. Survival is expressed as described for panel A (∗, P < 0.01). (C) Clonogenic assay of MEFs expressing wild-type Ku70 (WT), Ku70 S155A, and empty pMSCV vector (KO) compared to Ku70 bearing the phosphomimetic S155D substitution. Survival is expressed as described for panel A. WT, S155A, and pMSCV are significantly different from each other at all time points (P < 0.05), but asterisks to indicate that significance were omitted for clarity. Significance is indicated for S155D compared to pMSCV (∗, P < 0.001). (D) Western blot analysis of Ku subunit expression in Ku70^−/− MEFs expressing empty vector (pMSCV), wild-type Ku70 (WT), or Ku70 mutants as indicated. (E) Western blot analysis of phospho-ATF2 (69/71) in Ku70^−/− MEFs expressing wild-type Ku70 (WT), Ku70 S155A, and Ku70 S155D or empty vector (pMSCV). Cells were either left untreated (no IR) or subjected to 10 Gy and collected 2 h later. Western blot analysis was done with a phospho-ATF2 69/71 antibody (p69/71), an ATF2 antibody to determine total ATF2 protein expression, and actin as indicated.