Figure 5.

DBF4 is localized at centromeres throughout S-phase. (A) U2OS cells transiently expressing DBF4-FLAG-Strep were treated with 10 μM EdU for 15 min prior to pre-extraction and fixation. EdU-containing DNA is shown in magenta, DBF4 in green, and centromeres (ACA) in red. Early, mid or late S-phase cells were determined according to the pattern of EdU incorporation. Areas indicated in boxes are magnified next to the respective images. Scale bar = 10 μm. (B) U2OS cells were transfected with plasmid expressing EGFP-DBF4 fusion protein and analysed by fluorescence microscopy. EGFP signal is in green and ACA centromeric staining in red. (C) Chromatin immunoprecipitations (ChIP) were performed with anti-Strep and anti-RNA pol II (RPB1) antibodies from extracts of T-REx-EV and T-REx-DBF4 cells. The amounts of the non-centromeric GAPDH promoter and Chromosome 1 centromeric (Cent1) DNA recovered were determined by qPCR relative to the input samples. Bars represent the Mean ± S.D. of three technical replicates. Results are representative of three independent experiments.