MTOC-noncompetent centrioles are unable to support duplication. (A and B) Plk1^as cells induced to form de novo centrioles were treated with 3MB-PP1 or monastrol in late G2 for 3 h, treated with RO-3306 for 2 h to cause mitotic exit, incubated for 10 h to allow S-phase entry, and pulse labeled with BrdU for 1 h followed by a 4-h chase. BrdU-positive cells containing multilobed nuclei were identified, and their freestanding centrioles were analyzed for duplication using the centriolar markers centrin (centrin::GFP), hSas-6, and C-Nap1. A duplicated centriole pair is defined as a centrin doublet that is hSas-6 positive (marking newly formed daughter centrioles) and C-Nap1 positive (marking mother centrioles). Note that although centrin labels all centrioles, at some viewing angles, immature daughter centrioles containing small amounts of centrin may be blocked by mother centrioles and, therefore, not visible. In control cells (monastrol treated), most centriole pairs were labeled with hSas-6 and C-Nap1 (arrows), although a few of them were viewed as single centrin foci. Nevertheless, because all these centrioles lost hSas-6 labeling in early G1 (Fig. 4 E), the regaining of hSas-6 signal in S phase indicates that they had initiated duplication. Conversely, in 3MB-PP1–treated cells, almost all of the freestanding centrioles are centrin singlets. These centriole singlets have hSas-6 labeling and lack C-Nap1 labeling, indicating that unmodified centrioles are unable to support duplication. Centriole rosettes in 3MB-PP1–treated cells containing C-Nap1–labeled mother centrioles are shown in the insets. (C) Quantification of centriole duplication after down-regulation of Plk1 or Eg5. Numbers of centrioles are indicated. Error bars indicate standard deviations.