Fig. 2.

G₁ arrested CHO cells support centrosome duplication. A: Maximum intensity projection of centrin-GFP fluorescence in a live CHO-A8 cell, released from G₀ in the presence of 600 mM mimosine for 20 h. There are two pairs of bright fluorescent dots, as well as centrin-GFP fluorescence in the nuclear region. B: Corresponding DIC image of cell in (A). C,D: Immunofluorescent images of CHO-A8 cells arrested in G₁ for 24 h. Cells are labeled with anti-γ tubulin/centrin 2-GFP, showing two pairs of centrin-GFP fluorescent dots, co-incident with anti-γ tubulin foci. E: Transmission electron micrograph of a CHO-A8 cell released from G₀ into mimosine for 20 h prior to fixation and embeddment. Each of the parental centrioles is assembling a short pro-centriole at right angles. F: Centriole number in a population of G₀ cells, and G₁ cells (24, 48, and 72 h after serum release into mimosine). Graph shows percentage of cells containing either 2, 4, or >4 centrioles per cell. Data are average of three experiments, 200 cells per condition. G: Centriole number in cells treated with mimosine or HU fixed at intervals following serum release from G₀. Note that the number of cells with four centrioles begins to increase between 14 and 16 h for mimosine, and 9 h for HU. Average of three experiments, 200 cells per time point. H–H″/I–I″: Immunofluorescence images of two separate CHO-K1 cells, released from G₀ into mimosine for 20 h, then fixed and labeled with anti-γ tubulin (H,I), and anti-centrin 2 (H′,I′).H″, I″: Merged images, showing co-localization of centrin pairs and γ-tubulin positive centrosomes. Each cell contains a pair of anti-centrin 2 positive foci coincident with the two γ-tubulin positive foci. J: BrdU incorporation in control versus cell cycle arrested cells. Graph shows % of cell positive for anti-BrdU fluorescence. Mimosine arrests cells prior to the onset of S-phase. K: Flow cytometry of control, and mimosine treated CHO-A8 cells. DNA content of mimosine-treated cells following drug wash-out as they progress into S-phase over 24 h. Bar in (B) = 10 µm, in (D) = 10 µm, and in (E) = 0.5 µm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]