Lack of ATM kinase activity and genomic instability in ATM−/− fibroblasts. (A) Western blots from heart, liver, cerebellum and cerebrum tissues harvested from ATM+/+, ATM+/−and ATM−/− pigs. A slightly reduction of the ATM KD band (∼370 kDa) was seen in ATM+/−pigs; the band was completely absent in tissue derived from pigs ATM−/− (B) Western blotting on three independent fibroblast lines established from ATM+/+, ATM+/− and ATM−/− skin biopsies showed similar reduced levels of KD. (C) ATM kinase activity in fibroblast lines exposed to ionizing irradiation at 6 Gy. Increased phosphorylation of ATM substrates (top blot) and p53 (Ser15) and Chk2 (Thr68) (middle blot) were seen after 0 and 4 h (T0 and T4) of 6 Gy exposition in ATM+/+, ATM+/− fibroblasts while no increased signal was detected in ATM−/− fibroblasts. (D) Cytogenetic analysis of chromosomal breaks in fibroblasts showed an increase in chromosomal breakage (arrowheads) in cells derived from ATM−/− pigs.