Figure 3.

Depletion of Cdc6 in S phase results in prolonged DNA synthesis but inhibited new origin firing. HeLa cells grown on coverslips were synchronized at early S phase and then transfected with luciferase or Cdc6 endonuclease-prepared small interfering RNA as in Fig 2. (A) Luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA)-transfected cells were pulse-labelled with 10 mM 5-bromo-2′-deoxyuridine (BrdU; 20 min), fixed at hourly intervals and immunostained with an anti-BrdU antibody. The percentages of BrdU-positive cells were scored (>300 cells) at the indicated time points using a fluorescence microscope. (B) At 5 and 10 h after transfection, cells were labelled sequentially with 10 μM 5-chloro-2′-deoxyuridine (CldU) followed by 15 μM 5-iodo-2′-deoxyuridine (IdU; 10 min pulse each). DNA fibres were immunostained with anti-CldU and anti-IdU antibodies. CldU/IdU-positive DNA fibres (>150) were visualized and quantified using a fluorescence microscope. Expression levels of Cdc6 at the respective time points were determined by immunoblot analysis.