Cell cycle behavior of appendage proteins in primary HSPCs. (A) Centrioles and centrosomes are asymmetric. At G1 phase, the sole centrosome is composed of an M-centriole (1) and a D-centriole (2). At the beginning in S phase and extending in the G2 phase, two new D-centrioles (3 and 4) are generated. The D-centriole (2) acquires appendage proteins later in the cell cycle, whereas the newly formed D-centrioles (3 and 4) are devoid of appendages. Consequently, the centrosome containing the grandmother centriole (1) is the older centrosome in mitosis. The bar indicates the inter-centrosomal distance (d) in G2 phase. (B) Representative immunofluorescence images showing the localization of the DA protein Cep164 at the centrosome in different cell cycle phases in RPE1 cells. γ-tubulin was used as a centrosome marker. The insets represent magnifications of the centrosome signals. Nuclear pore complex antibody Mab414 was used for nuclear envelope staining. Cep164 release from the centrosome starts before nuclear envelope breakdown (NEB; G2/Pro). Scale bars, 20 µm (panel) and 2 µm (inset). (C) Quantification of cell cycle–dependent behavior of DA proteins and ODF2 in primary HSPCs. Appendage proteins were labeled with specific antibodies. γ-tubulin serves as a centrosome marker. The levels of the indicated appendage protein were measured at each centrosome (centrosome 1 and centrosome 2) during interphase (inter), G2, prometaphase (pro), metaphase (meta), anaphase (ana), and telophase (telo) and normalized to the average of interphase (marked by gray shading). Graphs depict fluorescence average intensity in a.u. Graphs show average ± SD of at least two independent experiments. Graphs represent mean ± SD of at least two independent experiments. Numbers below the bars represent total number of cells analyzed for each condition. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.