Daughter centrioles do not contribute to PCM recruitment. (A) Untreated (doublets) or hSas-6–depleted (singlets) RPE1 cells stably expressing centrin::GFP at indicated cell cycle stages were stained with antibodies against γ-tubulin. G2 cells were obtained by treatment with the Cdk1 inhibitor RO-3306, which arrested cells at G2/M. Mitotic cells were enriched by RO-3306 washout and identified by DAPI staining. Insets show a higher magnification of centrosomes. (B) Higher magnification of a pair of centrosomes from a G2/M cell extracted with Pipes buffer before fixation and stained for centrin and γ-tubulin. Arrowheads indicate weak centriolar γ-tubulin associated with daughter centrioles (arrows). (C) Quantification of γ-tubulin signals associated with centrosomes in different cell cycle stages. Numbers of centrosomes are indicated. Error bars indicate standard deviations. (D) Electron micrographs of mitotic cells. We obtained random sections of >20 mitotic centrosomes. Two representatives are shown here, one pair of centrioles from each cell. Mother (arrowheads) and daughter (arrows) centrioles are shown in both cross and longitudinal sections. Note that most of the microtubules and electron-dense material associate with mother centrioles.