Figure 6

Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser³⁴⁵phosphorylation and G2 arrest in HeLa cells. (A) a. Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b. Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. (B) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser³⁴⁵ antibody (a). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls (b). (C). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.