FIG. 4.

After silencing of p60, proliferating cells do not accumulate in S phase before cell death. (A) Cell cycle analysis. HeLa cells were transfected with nontarget (nt) and p60 siRNAs as indicated. Untransfected cells were used as a control. At 36 h posttransfection, nuclear DNA content was analyzed by flow cytometry. Identical results were obtained at 24 h posttransfection. (B) Quantitative analysis of DNA replication. Nuclei from the cells shown in panel A at 24 h posttransfection were incubated in their cognate cytosol in vitro to label active DNA replication foci (see Materials and Methods) (17). The percentages of nuclei with active DNA replication foci were determined by confocal fluorescent microscopy (see below). (C) Representative fields of replicating nuclei. Replication reactions were performed as indicated, and nuclei were analyzed by confocal fluorescence microscopy. Nuclear DNA was counterstained with propidium iodide (red signal) and replicated DNA was counterstained with fluorescein-conjugated antidigoxigenin Fab fragments (green signal). Merged images are presented, showing sites of replicated nuclear DNA in yellow and nonreplicating nuclei in red.