Journal: Oncogene

Article Title: Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex.

doi: 10.1038/s41388-021-02106-8

Figure Lengend Snippet: Fig. 1 DAB2IP prevents premature mitotic exit and maintains MCC stability. DAB2IP-proficient and -deficient C4-2 (C4-2 D2 and C4-2 Neo cells) (A) and PC3 (PC3 siCtrl cells and siD2) (B) cells were synchronized in prometaphase using nocodazole (50 ng/ml) and then released into fresh media. Cells were collected at the indicated times after release. The expression of DAB2IP, Cyclin B1, Securin, and HSP70 were detected via immunoblotting. Cdc20 and APC/C complex were immunoprecipitated from nocodazole-blocked C4-2 D2 and Neo (C), and from DAB2IP- knockdown PC3 and its control (D) prometaphase cells lysates. The amounts of Mad2 and BubR1 binding with Cdc20 and Cdc27 were determined by immunoblotting.

Article Snippet: The HA-tagged and Myc-tagged Cdc20 plasmids were obtained from Addgene (Addgene, #11594 and #11593) [50].

Techniques: Expressing, Western Blot, Immunoprecipitation, Knockdown, Control, Binding Assay

Journal: Oncogene

Article Title: Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex.

doi: 10.1038/s41388-021-02106-8

Figure Lengend Snippet: Fig. 2 DAB2IP interacts with Cdc20, and inhibits the ubiquitylation mediated degradation of Cdc20 in prometaphase. A, B C4-2 D2 cells were synchronized at prometaphase and the mitotic cells were collected by the shake-off method. Cells lysates were immunoprecipitated with anti-DAB2IP (A) or anti-Cdc20 (B) or IgG antibodies, and the interaction with Cdc20, DAB2IP, Cdc27, BubR1, and Mad2 were analyzed by immunoblotting. C Schema of different truncated domains of DAB2IP. D, E Various truncated cDNA constructs of DAB2IP were co-transfected with HA-Cdc20 into HeLa cells. HeLa cells were then synchronized at prometaphase and the mitotic cells were collected by the shake-off method. Cells lysates were immunoprecipitated with anti-Flag with antibody. The signal of HA and Flag were determined by immunoblotting. F Myc-Cdc20 and HA-Ubi were co-transfected with Flag-DAB2IP or empty vector in HeLa cells. Cells were harvested at 6 h after MG132 (10 μM) treatment. Cdc20 was immunoprecipitated using anti-Myc antibody. Co-IP products were analyzed by immunoblotting using anti-HA and anti-Myc antibodies. Equal loading of whole cell lysates were subjected to immunoblotting by anti-Flag antibody. G C4-2 D2 and Neo cells were arrested at prometaphase and treated with cycloheximide (0.1 mg/ml) and nocodazole (50 ng/ml) for the indicated times. The degradation rate of Cdc20 was determined by immunoblotting. H Mean Cdc20 protein levels in cycloheximide-treated C4-2 D2 and Neo cells (n = 4; error bars, s.e.m.). The intensity was normalized to HSP70 levels.

Article Snippet: The HA-tagged and Myc-tagged Cdc20 plasmids were obtained from Addgene (Addgene, #11594 and #11593) [50].

Techniques: Immunoprecipitation, Western Blot, Construct, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex.

doi: 10.1038/s41388-021-02106-8

Figure Lengend Snippet: Fig. 4 DAB2IP-deficient prostate cancer cells are sensitive to Mps1 inhibitor. A Paired C4-2 cells were exposed to indicated concentrations of AZ3156, a specific Mps1 inhibitor, for 48 h and MTT assay was performed. Results obtained were from three independent experiments (means ± SD; ***P < 0.001 and *P < 0.05 as compared with Neo cells). B 1 × 104 cells were seeded in 35 mm2 dishes at day 0, cells were treated with or without AZ3156 (0.5 μM) and cells were counted to determine cell proliferation rates at indicated days (1, 2, and 3 days) (means ± SD; *P < 0.05, ***P < 0.001 as compared with untreated cells). C C4-2 D2 and Neo cells were arrested at prometaphase and treated with 0.5 and 1 μM of AZ3156 along with nocodazole for an additional 2 h. APC/C complex was immunoprecipitated from C4-2 D2 and Neo cells lysates treated or untreated with Mps1 inhibitor. The amounts of Mad2, BubR1, and Cdc20 binding with Cdc27 were determined by immunoblotting. D C4-2 Neo and D2 cells were treated with 10 μM MG132 for 4 h, then treated with or without 0.5 μM AZD3156 along with MG132 for an additional 2 h. Chromosome alignment was determined by immunofluorescent staining using anti-α-tubulin and Crest antibody; DNA was visible by DAPI. Scale bars are 5 μm. E Proportion of cells with chromosome-missegregation and misalignment in C4-2 Neo and C4-2 D2 cells treated with or without AZD3156 are presented as the mean and SD from three independent experiments (**P < 0.01, ***P < 0.001 as compared with Neo cells). F Cells were exposed to 1 μM of AZD3156 for 24 h and subjected to immunoblotting with anti-PARP, anti-DAB2IP, and anti-Actin antibodies.

Article Snippet: The HA-tagged and Myc-tagged Cdc20 plasmids were obtained from Addgene (Addgene, #11594 and #11593) [50].

Techniques: MTT Assay, Immunoprecipitation, Binding Assay, Western Blot, Staining

Journal: Oncogene

Article Title: Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex.

doi: 10.1038/s41388-021-02106-8

Figure Lengend Snippet: Fig. 6 Phosphorylation of DAB2IP at its Thr531 and Thr546 sites activates the PLK1-Mps1 signal pathway and inhibits Cdc20 ubiquitylation in prometaphase. A Flag-tagged-DAB2IP, -DAB2IP 2A (T531A/T546A), -DAB2IP 2D (T531D/T546D), empty vector and HA-PLK1 were transfected into HeLa cells. The cells were then treated with 50 ng/ml nocodazole for 16 h and mitotic cells lysates were immunoprecipitated with anti-Flag antibody; the HA signal was examined by immunoblotting. B Immunoblotting analysis levels of PLK1- pT210, PLK1, Mps1, BubR1 in mitotically arrested or asynchronous PC3 cells with siRNA-mediated DAB2IP suppression and overexpression of siRNA-resistant DAB2IP (rWT), siRNA-resistant DAB2IP 2A (r2A), and siRNA-resistant DAB2IP 2D (r2D) constructs. C PC3 cells with siRNA- mediated DAB2IP suppression and overexpression of siRNA-resistant DAB2IP (rWT), siRNA-resistant DAB2IP 2A (r2A), and siRNA-resistant DAB2IP 2D (r2D) constructs were arrested in mitosis by nocodazole, and shake-off cell lysates were immunoprecipitated by anti-Mps1 antibody. The phosphorylation of Mps1 on its Thr676 site and the amount of Mps1 protein were detected. D Flag-tagged-DAB2IP, -DAB2IP 2A (T531A/T546A), -DAB2IP 2D (T531D/T546D), empty vector and Myc-Cdc20 were transfected into HeLa cells. The cells were then treated with 50 ng/ml nocodazole for 16 h and mitotic cells lysates were immunoprecipitated with anti-Flag antibody; the Myc signal was examined by immunoblotting. E Myc-Cdc20 and HA-Ubi were co-transfected with Flag-DAB2IP, Flag-DAB2IP 2A (T531A/T546A), Flag-DAB2IP 2D (T531D/ T546D) or empty vector in HeLa cells. Cells were harvested at 6 h after MG132 (10 μM) treatment. Cdc20 was immunoprecipitated by using anti-Myc antibody. Co-IP products were analyzed by immunoblotting using anti-HA and anti-Myc antibodies. Equal loadings of whole cell lysates were subjected to immunoblotting by anti-Flag and anti-HA antibodies. F The intensity of ubiquitinated proteins was normalized to VC transfected cells, then graphed for four independent experiments (n = 4; ***P < 0.001 as compared with VC transfected cells.).

Article Snippet: The HA-tagged and Myc-tagged Cdc20 plasmids were obtained from Addgene (Addgene, #11594 and #11593) [50].

Techniques: Phospho-proteomics, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Over Expression, Construct, Co-Immunoprecipitation Assay