Figure 7.

Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser³⁴⁵ phosphorylation and G2 arrest in CEM-SS cells. (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. (B) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser³⁴⁵ antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. (C). CEM-SS were treated the same way as described in (B). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.