Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: Clinicopathological features of 232 patients with thyroid cancer.

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Expressing, Dissection

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: BUB1 expression with its clinical significance in TC. (A) Association between PFS and BUB1 expression (RNA‐seq) in TC from Kaplan–Meier plotter database. (B) Correlation between BUB1 expression (IHC) and PFS based on a cohort of a total of 232 samples of PTC from tissue microarrays. (C, D) BUB1 expression in normal and TC samples and the relevance with tumour stage in GEPIA database. (E–G) Expression of BUB1 in TC from datasets GSE33630, GSE29265 and GSE65144. (H) BUB1 expression was examined in normal, PTC and ATC tissues through immunohistochemistry staining. (I) BUB1 protein levels among TC cells were detected using WB assays. (* p < 0.05, ** p < 0.01, *** p < 0.001; mean ± standard deviation (SD)).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Expressing, RNA Sequencing, Immunohistochemistry, Staining, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of BUB1 expression with patient prognosis.

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Expressing, Dissection

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: Reduced expression of BUB1 in TC cells suppresses invasive phenotype. (A, B) Verification of BUB1‐siRNA silencing efficacy at mRNA and protein levels. Cell viability assays (C) and colony‐formation assays (D) were measured after BUB1 silencing. (E) The cell cycle of BUB1 silencing in TC cells was determined using flow cytometry. (F–H) Invasion, migration and wound healing assays of BUB1 silence were conducted in TC cells. (* p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Expressing, Flow Cytometry, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: Nude mice with ATC xenografts exhibit decreased tumorigenesis and slower tumour growth after BUB1 knockdown. The xenografts were developed from BUB1‐WT or BUB1‐KD 8505C cells. (A–E) Body weight, tumour formation rate, tumour weight and tumour volume. (F) H&E staining of tumour tissues. (G) Detection of Ki67 expression performed by immunohistochemistry. (H) The zebrafish xenograft model was employed to evaluate the metastatic ability of 8505C cells, after BUB1 knockdown. (I) Quantification of disseminated tumour foci. (* p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Knockdown, Staining, Expressing, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: The effect of tumour inhibition by BUB1 inhibitor BAY‐1816032 in vitro. (A) We exposed cancer cells to varying BAY‐1816032 concentrations (0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 μM) for 48 h. CCK‐8 assays were employed to investigate cell viability. Colony‐formation assays (B), cell cycle (C), invasion (D), migration (E) and wound healing assays (F) were detected after being treated with BAY‐1816032 in a dose‐dependent manner (0, 2.5, 5 μM). (* p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Inhibition, In Vitro, CCK-8 Assay, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: The activity of BUB1 inhibition by BAY‐1816032 for tumour growth in ATC in vivo. (A) A BAY‐1816032 oral administration anti‐tumour experiment performed with a mouse xenograft model is shown. We divided the xenografts into the control group and the BAY‐1816032 group after 2 weeks. (B–E) Body weight, tumour weight and tumour volume. (F–G) H&E staining and immunohistochemistry of Ki67 in each group of mice tumour tissues. (H) H&E staining in each group of mice hearts, livers, spleens, lungs and kidneys. (* p < 0.05, ** p < 0.01; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Activity Assay, Inhibition, In Vivo, Control, Staining, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: BUB1 evokes CIN in ATC. (A) A biological process enrichment GO annotation was performed using GSEA grouped by expression of BUB1. (B, C) Gain or loss of the chromosomal segments in 8505C cells after BUB1 knockdown was detected by WES. (D) Immunofluorescent staining of α‐tubulin to detect the formation of abnormal spindle after BUB1 knockdown. (E) The nuclear abnormalities in BUB1‐KD 8505C cells, as revealed by the Giemsa staining, include nucleoplasmic bridges (NPB), micronucleus (MN) and nuclear buds (NBUD). (** p < 0.01, *** p < 0.001; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Expressing, Knockdown, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: BUB1 promotes CIN through interacting with KIF14 in ATC. (A) All significantly enriched gene ontology‐biological process terms from the top 10 enriched genes between high and low BUB1 expression were displayed on a heatmap. KIF14 expression (B–D) and its clinical significance (E) in TC. (F) Correlation analysis of BUB1 and KIF14 expression levels in the GEPIA database. (G) Immunofluorescence assays were conducted to verify the colocalization of BUB1 and KIF14. (H) Lysates derived from Nthy‐ori 3‐1 and 8505C cells were utilized for co‐IP of BUB1 and KIF14. The odds ratio of KIF14/BUB1 protein expression was calculated as KIF 14 IP KIF 14 input ÷ BUB 1 IP BUB 1 input . (I) Verification of KIF14 siRNA silencing efficacy at the mRNA level in 8505C cells. Immunofluorescent staining (J), Giemsa staining (K), and cell cycle distribution analysis (L) were performed after silencing of KIF14. (M) Temporal variation in p‐H3 examined by WB. (* p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Expressing, Immunofluorescence, Derivative Assay, Co-Immunoprecipitation Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability

doi: 10.1111/jcmm.18182

Figure Lengend Snippet: BUB1 induces CIN of ATC through phosphorylation of KIF14 at Ser 1292 . (A, B) The effects of BUB1 knockdown and BUB1 inhibitor BAY‐1816032 on KIF14 phosphorylation were detected using Phos‐tag assays. SDS‐PAGE gels were used to conduct WB for total KIF14. The expression level of p‐KIF14 protein was determined by its ratio to the total expression level of KIF14 protein. (C) The potential phosphorylation sites of KIF14 were retrieved from the UniProt website and mutated to alanine (Ala), respectively. (D) The effects of Flag‐tagged KIF14 mutant plasmids on its phosphorylation were conducted by a Phos‐tag assay in 8505C cells. (E) Cell viability assays were measured after transfecting with Flag‐tagged KIF14 mutant plasmids. (F) Lysates derived from 293T cells were utilized for co‐IP of HA‐BUB1 and FLAG‐KIF14. The cells were all transfected with HA‐BUB1 but were transfected with KIF14 ΔSer1292 mutant or WT plasmids, respectively. The odds ratio of FLAG‐KIF14/HA‐BUB1 protein expression was calculated as FLAG − KIF 14 IP FLAG − KIF 14 input ÷ HA − BUB 1 IP HA − BUB 1 input . (G) Lysates derived from 293T cells were utilized for co‐IP of HA‐BUB1 and FLAG‐KIF14. The cells were first transfected with HA‐BUB1 and FLAG‐KIF14 WT plasmids and then treated with BAY‐1816032 in a dose‐dependent manner. The odds ratio of FLAG‐KIF14/HA‐BUB1 protein expression was calculated as FLAG − KIF 14 IP FLAG − KIF 14 input ÷ HA − BUB 1 IP HA − BUB 1 input . (H) The in vitro kinase assays. BUB1 kinase was mixed with purified recombinant KIF14 or KIF14 ΔSer1292 protein, and KIF14 phosphorylation was examined by Phos‐tag assays. The expression level of p‐KIF14 protein was determined by its ratio to the total expression level of KIF14 protein. Immunofluorescent staining (I), Giemsa staining (J) and cell cycle distribution analysis (K) were performed after overexpression of KIF14 ΔSer1292 mutant (* p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SD).

Article Snippet: For WB, the primary antibodies included: BUB1 (A18053, ABclonal, Wuhan, China), KIF14 (26000‐1‐AP, Proteintech), p‐H3 (53,348, CST, USA), Flag (AE005, ABclonal), HA (AE105, ABclonal), GAPDH (10494‐1‐AP, Proteintech).

Techniques: Phospho-proteomics, Knockdown, SDS Page, Expressing, Mutagenesis, Derivative Assay, Co-Immunoprecipitation Assay, Transfection, In Vitro, Purification, Recombinant, Staining, Over Expression

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: HeLa cells were transiently transfected with a control siRNA or two independent siRNA oligos (Bub1 si5 and si6) against Bub1 and the following experiments were conducted: (A) Total cell lysates were harvested 36 hours after transfection followed by immunoblotting with the indicated antibodies. (B) Cells were treated with 4Gy of IR. At 24 or 48 hours after IR, cells were stained with the MTT-based proliferation assay. Shown are the averages of relative survival rates, and * indicates statistical significance (P=0.001, T-test). (C) Cells were treated with indicated doses of IR and colony formation assays were conducted. Shown are the averages of at least triplicate samples. Standard errors are shown by error bars. (D) Cells were plated onto cover-slips and after IR cells were fixed and stained with the anti-Histone λ-H2AXantibody followed by immunofluorescence microscopy. Nuclear H2AX foci were counted and shown are the averages of 100 cells. Standard errors are shown by error bars. Statistical analyses were conducted using T-test. (E) The Single Cell Gel Electrophoresis assay was conducted and Olive Tail Moment was recorded. Shown are the averages of 100 cells. Standard errors are shown by error bars.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transfection, Control, Western Blot, Staining, Proliferation Assay, Immunofluorescence, Microscopy, Single Cell Gel Electrophoresis

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: (A) HeLa cells transfected with control or Bub1 siRNA were treated with mock or IR (4Gy). Total cell lysates were collected followed by immunoblotting using indicated antibodies. (B) HeLa cells stably transfected with control shRNA or ATM shRNA were treated with mock or IR (4Gy). Total cell lysates were collected followed by immunoblotting using indicated antibodies.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transfection, Control, Western Blot, Stable Transfection, shRNA

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: Total cell lysates were harvested from: (A) HeLa cells treated with mock or IR (4Gy); (B) Camptothecin (CPT); (C) HeLa cells treated with mock or IR (4Gy) in the presence or absence of KU55933; (D) SV-40 transformed fibroblast cells lines GM0637 and GM9607 treated with mock or IR (4Gy); (E) HeLa cells transiently transfected with either control siRNA or ATM siRNA followed by mock or IR treatment. Western blot analyses were conducted using indicated antibodies. and (F) The in vitro kinase assay using ATM fragments (either GST-tagged N-terminal a.a. 248-522 or C-terminal fragments of ATM a.a. 2709-2964) in the presence of non-phosphorylated form of Bub1 peptides. The blot was stained by coomassie blue and immunoblotted with the anti-phospho-Ser314 Bub1 antibody.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transformation Assay, Transfection, Control, Western Blot, In Vitro, Kinase Assay, Staining

Journal: DNA repair

Article Title: The kinetochore protein Bub1 participates in the DNA damage response

doi: 10.1016/j.dnarep.2011.10.018

Figure Lengend Snippet: (A) HeLa cells were transiently transfected with vector-only, wild-type, or S314A mutant of Bub1 followed by experiments Western blot analysis to detect Histone H2A Thr121 phosphorylation in response IR; (B) HeLa cells transiently transfected with vector-only, wild-type, S314A or S314E mutant of Bub1 followed by immunoblotting with indicated antibodies. (C) The cell survival assay after IR in HeLa cells transfected with vector, wild-type or S314A Bub1; and (D) the single cell gel electrophoresis assay to detect comet tail formation in HeLa cells transfected with vector, wild-type or S314A Bub1.

Article Snippet: The control siRNA for Bub1 knock-down was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the two Bub1 siRNAs (Bub1-5si,CGAAGAGUGAUCACGAUUU; Bub1-6si, CAAAGAAGGGUGUAAACA) were purchased from Thermo Scientific (Rockford, IL). siRNAs were transfected into cells by oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction and 20nM siRNAs were used in each experiment.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Phospho-proteomics, Clonogenic Cell Survival Assay, Single Cell Gel Electrophoresis