Fig. 5. Downregulation of B-Myb by RNA interference leads to a defect in S phase and the nuclear localisation of DNA replication. (A) Flow cytometric analysis of siRNA-transfected HEL cells cultured in the presence of TPA for 64 hours followed by 16 hours in the presence of TPA and BrdU. Incorporation of BrdU was detected by immunofluorescence using a FITC-conjugated mouse anti-BrdU monoclonal antibody and FITC-conjugated mouse IgG1 isotype control (FL1, vertical axis), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis). The histograms on the right show green fluorescence (FL1, horizontal axis) of 4C cells stained with a FITC-conjugated mouse anti-BrdU monoclonal antibody. The black-shaded histograms indicate the extent of anti-BrdU staining, whereas the grey lines show the staining obtained with the isotype control. (B) Immunofluorescence analysis of siRNA-transfected HEL cells grown in the presence of TPA for 64 hours and then pulse-labelled with BrdU for 45 minutes. Incorporation of BrdU was detected by indirect immunofluorescence using a mouse anti-BrdU antibody and PE-conjugated goat anti-mouse Ig secondary antibody (red). Nuclei were stained with DAPI (blue). Upper panels: control siRNA. Middle panels: B-myb siRNA1. Lower panels: B-myb siRNA2. (C) Histogram representing the pattern of incorporation of BrdU in 100 cells treated with each of the siRNAs. NM, no match.