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Fig. 3. Centrosome replication in HU-arrested CHO cells. Cells were fixed at different time points during the experiment summarized in P. One to four centrosomes are shown in A-F. Particularly prominent is the centrosome surrounded by the prominent PCM (G). New daughter centrosomes can be seen emerging next to a larger mother centrosome (arrows in H,J). This frequently occurs in an asynchronous manner, resulting in the formation of highly heterogeneous numbers of centrosomes in cells treated with HU for extended periods of time (H-M). Cells shown in N and O include numerous cytoplasmic foci dispersed to varying degrees throughout the cell. Note the presence of GFP-centrin inside the nucleus at later stages of HU treatment (M-O). (P) Quantification of the frequency of CHO cells with one to two (blue), three to four (purple) and more than five (yellow) centrosomal dots as detected by centrin-tagged GFP fluorescence. Cells were synchronized in M phase and plated into a dish containing 2 mM HU (0 hours). Centrosomal dots were counted at different times during HU incubation, after extraction of cells with detergent-containing medium. More than 200 cells were counted for each sample. Bars, 5 µm (A-C,E-L); 10 µm (D,M-O); 1 µm (inset of D).
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