Fig. 5. Introduction of Cdt1 into quiescent Rat-1 cells induces chromosomal damage and activation of ATM-Chk2 without replication. Rat-1 cells were arrested in quiescence, then infected with lentiviruses carrying either wild-type or Cy mutant T7-Cdt1 or backbone vector, and subjected to analyses after 144 hours. (A) Whole-cell lysates were immunoblotted. Asynchronous (AS) CB4 cells serve as a positive control for T7-Cdt1 expression and ATM-Chk2 activation. (B) After further treatment with BrdU for 6 hours, cells were immunostained with anti-BrdU. Asynchronous (AS) Rat-1 cells serve as a positive control for BrdU uptake. (C) Cells were double-immunostained with anti-T7 and anti-phospho-ATM antibodies.