Journal: bioRxiv

Article Title: Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase to ensure proper centriole duplication cycle

doi: 10.1101/2020.05.10.086702

Figure Lengend Snippet: ( A ) Precocious centriole disengagement caused by G2/M phase arrest was suppressed by BI2536 (Plk1 inhibitor, 100 nM). HeLa cells were synchronized in the G1/S phase by Aphidicolin (1.2 μg/mL) for 17 h, then released into fresh medium for 4 h. Next, the cells were treated with DMSO (control), RO3306 (Cdk1 inhibitor, 10 μM), or both of RO3306 and BI2536 (100 nM) for 24 h. HeLa cells were immunostained with antibodies against centrin2 (green) and Cep192 (red). Scale bar, 5 μm. ( B ) Histograms represent frequency of the interphase cells with the indicated phenotype observed in (A). Values are mean percentages ± s.d. from three experiments (n = 50 for each experiment). ( C ) Precocious centriole disengagement in Cep57/Cep57L1-depleted cells was not suppressed by BI2536. HeLa cells were treated with siControl or siCep57/Cep57L1 for 24 h, followed by treatment of DMSO (control) or BI2536 (100 nM) for additional 24 h. The cells were immunostained with antibodies against centrin2 (green) and CENP-F (red). ( D ) Histograms represent frequency of cells in the G2 phase with the indicated phenotype observed in (C). Values are mean percentages ± s.d. from three experiments (n = 30 for each experiment). ( E ) BI2536 suppressed recruitment of PCNT at disengaged daughter centrioles. HeLa-GFP-centrin1 cells were treated as in (C), and immunostained with antibodies against GFP (green), CENP-F (red) and PCNT (cyan). White/black arrowheads indicate PCNT positive/negative centrioles, respectively. ( F, G ) Histograms represent frequency of cells with >2 PCNT or γ-tubulin foci among the G2 phase cells with disengaged or amplified centrioles. Values are mean percentages ± s.d. from three experiments (n = 30 for each experiment). ( H ) Schematic illustration of the results in Figure 3. The activity of Plk1 is required for the recruitment of PCM components at the disengaged daughter centrioles, but not for precocious centriole disengagement itself. All scale bars, 5 μm. Tukey’s multiple comparisons test was used in (D) against total value of centriole disengagement and >4 centrioles to obtain p value. Two-tailed, unpaired Student’s t-test was used in (F) and (G) to obtain p value. **p < 0.01; *p < 0.05; NS not significantly different (p > 0.05).

Article Snippet: The following chemicals were used in this study: nocodazole (Wako, 31430-18-9), RO3306 (SIGMA, SML0569), BI2536 (AdooQ, A10134) and SiR-DNA (Spirochrome, CY-SC007)

Techniques: Amplification, Activity Assay, Two Tailed Test