(A) PCV2-infected cells were collected at the indicated time points postinfection, and whole-cell lysates were prepared and subjected to SDS-PAGE followed by immunoblotting. The protein levels of phosphorylated ATM, ATR, and DNA-PK were analyzed. (B) Immunofluorescence analysis of PCV2 infection-induced ATM, ATR, and DNA-PK activations. At 48 h postinfection, the PCV2-infected cells were coimmunostained with anti-PCV2 ORF1 (green) and anti-p-ATM (Ser1981) (Red), anti-PCV2 ORF1 (green) and anti-p-ATR (Ser 428) (red), or anti-PCV2 ORF1 (green) and anti-p-DNA-PK (Thr2609) (red). Nuclei were visualized by DAPI staining. (C) Treatment with the specific inhibitors reduces PCV2-induced phosphorylation of ATM, ATR, and DNA-PK. PK15 cells were inoculated with PCV2 in the presence or absence of the specific inhibitor for ATM kinase (ATMi), ATM/ATR kinase (ATM/ATRi), or DNA-PK kinase (DNA-PKi). Cell lysates at 48 h postinfection were harvested and subjected to SDS-PAGE followed by immunoblotting with antibodies to ATM, ATR, and DNA-PK phosphorylated forms. (D) Treatment with the specific inhibitors reduces PCV2-induced phosphorylation of several DDR downstream targets. PK15 cells were inoculated with PCV2 in the presence or absence of the DDR kinase inhibitors. Cell lysates at 48 h postinfection were harvested and subjected to SDS-PAGE followed by immunoblotting with antibodies to Chk1, Chk2, and p53 phosphorylated forms as well as Ku80 and Ku70. β-actin was used as the loading control. p-, phosphorylated.