Phenotypic analyses about the cell cycle in Cep57/Cep57L1-depleted cells. (A) The number of old mother centriole was one, regardless of the phenotypes in Cep57/Cep57L1-depleted cells. HeLa cells were treated with siControl or siCep57/Cep57L1 and immunostained with antibodies against centrin (green) and ODF2 (red). (B) Histograms represent the frequency of the interphase cells with the indicated number of ODF2 foci observed in A. Values are mean percentages ± SD from two independent experiments (n = 50 for each experiment). (C) Quantification of the duration from anaphase onset to next mitotic entry. HeLa-GFP-centrin1 cells were treated with siControl or siCep57/Cep57L1 and visualized for live-cell imaging for 48 h (n = 25). (D) Cep57/Cep57L1 codepletion did not affect cell cycle progression. HeLa cells were treated with the indicated siRNAs and followed by flow cytometric analysis. (E) Cep295 was recruited to the disengaged centrioles. HeLa cells were treated with siControl or siCep57/Cep57L1 and immunostained with antibodies against centrin (green) and Cep295 (red). The insets in a–c are the magnified views of the corresponding regions in the low-magnification view. (F) PCNT was not recruited to disengaged daughter centrioles in the S phase. HeLa cells were treated with siCep57/Cep57L1 and immunostained with antibodies against PCNT (green), CP110 (red), and PCNA (cyan). (G) Histograms represent the frequency of cells with more than two PCNT-positive centrioles among cells with separate or more than four CP110 foci in S phase, G₂ phase, and mitosis. Values are mean percentages ± SD from two independent experiments (n = 50 for each experiment). (H) PCNT was not recruited to disengaged daughter centrioles immediately after centriole disengagement in control cells as in Cep57/Cep57L1-depleted cells. HeLa cells were immunostained with antibodies against PCNT (green) and CP110 (red). (I) Ectopic MTOC activity of precociously disengaged daughter centrioles in Cep57/Cep57L1-depleted cells. HeLa cells were treated with siControl or siCep57/Cep57L1 and followed by nocodazole treatment (10 µM) for 3 h. After nocodazole treatment, the cells were cold treated for 1 h, followed by 1-min incubation at 37°C and immunostaining with antibodies against EB1 (green) and CP110 (red). White arrowheads indicate centrosomes. All scale bars, 5 µm in the low-magnification view, 1 µm in the inset. The normal distribution of datasets was confirmed using the Kolmogorov–Smirnov test in C. A two-tailed, unpaired Student’s t test was used in C to obtain P value. NS, P > 0.05.