Fig 1: 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 2: CDK5RAP2 regulates spindle orientation.A Schematic depicting calculation of spindle angles. The horizontal distance (X µm) and the vertical distance (Z µm) between two spindle poles of metaphase cells were measured by acquiring Z-stack images. Spindle angle (a?), the angle between the spindle axis and the substrate plane, was calculated using an inverse trigonometric function. B Representative Z-stack images of metaphase cells. HeLa cells were fixed and immunostained with anti-a-tubulin and anti-?-tubulin antibodies. White arrows indicate the focal planes of two spindle poles in two different sections. Scale bars, 5 µm. The spindle angle of HeLa cells after control or CDK5RAP2 knockdown was quantified. Data are presented as means ± SD; *P < 0.05, two-tailed Student’s t-test.
Fig 3: CDK5RAP2 knockdown suppresses stemness of OSCC cells.A, B Equal numbers of control and CDK5RAP2-knockdown stable Cal27 cells A or HSC-3 cells B were seeded into ultra-low-attachment plates and incubated for 8 days, after which the spheres (diameter > 50 µm) were counted. Scale bars, 50 µm. C Correlation between CDK5RAP2 and HNSCC CSC markers. D Heatmap presenting gene-expression profiles of control and stable CDK5RAP2-knockdown cells. Gene expression was measured using RNA-sequencing, and GSEA was performed. E Control and stable CDK5RAP2-knockdown cells were inoculated subcutaneously into nude mice. The panel shows representative images of tumor sections stained with antibodies against ALDH1, SOX2, CD44, CD133, Notch1, EZH2, and CCND1. Image J was used to analyze the relative expression intensities of indicated CSC markers from isolated xenograft tumors. Scale bar, 100 µm. All data are presented as means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test.
Fig 4: 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 ß-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.
Fig 5: 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.
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