Fig 1: Sas-6 is required for the maintenance of centriole engagement and pericentriolar matrix integrity. A, experimental scheme for the generation of mitosis-arrested HeLa cells by Sas-6 knockdown. HeLa cells were arrested at metaphase by sequential treatment with 2.5 μM thymidine, 5 μM STLC, and 10 μM MG132 and simultaneously transfected with Sas-6 siRNA for 18 h. B, knockdown efficiency of Sas-6 siRNA. HeLa cells were treated as described in (A), harvested, and analyzed by immunoblotting using antibodies against Sas-6 and GAPDH. C, Sas-6 knockdown induces abnormal centriole disengagement and disordered pericentriolar matrix (PCM) during mitosis. HeLa cells were treated as described in (A), fixed with methanol, and analyzed by immunofluorescence using antibodies against PCNT, CDK5RAP2, γ-tubulin, and Centrin1. DNA was stained with DAPI. Arrowheads indicate cracked PCM proteins. The boxed areas in each main image are zoomed into the right panels with separated channels. Scale bars, 5 μm (large images) or 1 μm (inset images). D, quantitation of the number of cells with aberrantly separated centrioles in (C). Centrioles with a distance greater than 1 μm were counted as separated. Approximately 200 cells were counted per sample, and three independent experiments were conducted. The statistical data are expressed as the mean ± SD. ∗∗p < 0.01 (Student’s t test). E, schematic diagram illustrating that Sas-6 knockdown after daughter centriole assembly induces untimely centriole disengagement and disrupts PCM integrity. See also Figs. S1 and S2.
Fig 2: CDK5RAP2 downregulation inhibits OSCC cell migration.A, B Wound-healing assay was performed using Cal27 A and HSC-3 B stable cells. Representative images are shown. Ratios of wound closure at 48 h or 6 h were quantified. C Transwell migration assay was performed using stable cells. Representative images are shown. The migrated cell number per field was quantified. Data are presented as means ± SD; *P < 0.05, ***P < 0.001, two-tailed Student’s t-test. Scale bars, 200 μm.
Fig 3: CDK5RAP2 is upregulated in OSCC.A–C CDK5RAP2 expression in OSCC according to sample types A, cancer stages B, and nodal metastasis status C. D Kaplan–Meier survival analysis of TCGA-OSCC cohorts grouped by CDK5RAP2 expression level. E IHC staining of CDK5RAP2 in OSCC. Rectangular area is enlarged. CDK5RAP2 expression was quantified using Image J (n = 50). Scale bars, 20 μm. Data are presented as means ± SEM; ns no significance, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: CDK5RAP2 downregulation inhibits OSCC tumorigenesis.A Cal27 cells were infected with a recombinant lentivirus containing CDK5RAP2 shRNA (CDK5RAP2 RNAi) or negative-control shRNA (Control), and then lysates of stable cells were immunoblotted with the indicated antibodies. B Colony-formation assay was performed using control and stable CDK5RAP2-knockdown cells. Colonies in each well were counted. C shRNA-carrying lines of Cal27 cells were injected subcutaneously into nude mice. The image shows isolated tumors. Mice were weighed every 4 days after injection, and the isolated tumors were weighed. Data are presented as means ± SD; ***P < 0.001, two-tailed Student’s t-test.
Fig 5: CDK5RAP2 expression is regulated by the Wnt signaling pathway.A, B HOK cells were treated with 25 μM ICG-001 A or 100 ng/mL Wnt3a B for 24 h and then cell lysates were immunoblotted with the indicated antibodies. CDK5RAP2 protein levels were quantified and normalized to the β-actin protein levels. C ChIP assay was performed using rabbit anti-CBP antibody or normal rabbit IgG. RPE1 cells were treated with DMSO as mock treatment or 25 μM ICG-001. D Schematic of predicted CBP-binding sites in the cdk5rap2 promoter region. Binding between CBP and the cdk5rap2 promoter was determined using a dual-luciferase reporter assay. RPE1 cells transfected with distinct promotor fragments were treated with or without ICG-001. Firefly luciferase intensity was normalized relative to Renilla luciferase intensity. Data are presented as means ± SD; ns no significance, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test.
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