Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication
Figure 7
Down-regulation of Orc1 inhibits DNA replication and enhances E2F1 recruitment to origins of DNA replication.
(A) Western blotting using the indicated antibodies at 72 h after treatment of U2OS cells with siRNAs against Orc1 or luciferase (Luc) control. (B) Flow cytometry profiles of U2OS cells treated for 72 h with the indicated siRNAs. The histogram on the right side shows the distribution of the cells in the different phases of the cell cycle; the reduction in the number of S-phase cells after Orc1 silencing is indicated by an arrow. (C) Flow cytometry profiles simultaneously showing detection of DNA content (propidium iodide staining) and BrdU incorporation (anti-BrdU antibody) at 72 h after RNAi. The dashed boxes indicate BrdU positive, S-phase cells. The histogram on the right side reports the percentage of S-phase/BrdU positive cells (mean±sem, indicated by error bars) of three different experiments. The asterisk (*) indicates significant statistical difference between ORC depletion experiments and luciferase control experiments. (D) Results of ChIP experiments performed in U2OS cells at 72 h after siRNA silencing of Orc1. The histograms show the quantification of origin-specific, cross-linked and immunoprecipitated DNA for the Lamin B2 origin after immunoprecipitation using the antibodies shown below each bar pair. The results are expressed as fold of enrichment of the specific origin sequence (B48) over a neighbouring control sequence (B10), as shown in Fig. 1A. The means±sem of at least three different experiments are shown. The asterisk (*) indicates statistically significant differences between ORC depletion experiments and luciferase control experiments. (E) Results of ChIP experiments performed by analyzing protein binding to the GM-CSF Ori1 and Ori2 origins in U2OS cells at 72 h after siRNA silencing of Orc1. The results are expressed as in (D), by showing the fold of enrichment of the specific origin sequences (#17 and #23) over a neighbouring control sequence (#21), as schematically represented in Fig. 1C.
