Journal: BMC biology

Article Title: Evolutionary conserved relocation of chromatin remodeling complexes to the mitotic apparatus.

doi: 10.1186/s12915-022-01365-5

Figure Lengend Snippet: Fig. 7 Inhibition of Aurora B kinase activity with ZM447439 and Barasertib affected the localization of BAF53a and Tip60 to the midbody. A Treatment with ZM447439: from left to right: DAPI (blue), anti- α-tubulin (green), tested protein (red), and merge. Anti-MKLP1 and anti-Aurora B immunostaining were used as positive and negative controls, respectively. Scale bar = 10 μm. The localization pattern of BAF53a and Tip60, as well as that of MKLP1 were affected, while no effect was seen for SRCAP and Aurora B. B The effects found with ZM447439 treatment are summarized in the graph. At least 300 telophases were scored in three independent experiments for both treated cells and control HeLa cells. C To test the effectiveness of ZM447439 inhibition, phosphorylation of the histone H3 (target of Aurora B kinase) during mitosis was evaluated; α-tubulin was used as loading control. D The effects found with Barasertib treatment are summarized in the graph. At least 200 telophases were scored in two independent experiments for both treated cells and control HeLa cells. A slight effect on Aurora localization was seen at the limit of significance values. E To test the effectiveness of Barasertib inhibition, phosphorylation of the histone H3 (target of Aurora B kinase) during mitosis was evaluated; α-tubulin was used as loading control. *=P<0.05, **<=P<0.005, ***=P<0.0005

Article Snippet: Asynchronous HeLa cells were treated for 35 min with the Aurora B inhibitor ZM447439 (SignalChem, A31901-01) and Barasertib (AZD1152-HQPA, Selleckchem, S1147) at a final concentration of 5 μM and 200 nM, respectively, and then fixed and stained.

Techniques: Inhibition, Activity Assay, Immunostaining, Control, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: SUMO2 modification of Aurora B and its impact on follicular development and atresia in the mouse ovary

doi: 10.3892/ijmm.2018.3541

Figure Lengend Snippet: Aurora B impacts the viability of granulosa cells. (A) The cell cycle of granulosa cells was assessed by FACS after the cells were co-cultured with ZM447439 at different concentrations for 48 h. (B) The proportion of each phase of the cell cycle. (C) Proliferation of the granulosa cells was assayed by WST-1. (D) A statistical analysis of the apoptosis rate. Each experiment was repeated three times, and the values are presented as the mean ± standard deviation; * P<0.05, ** P<0.01 and *** P<0.001 cf. the 0 μ M data. (E) The apoptosis rate of the granulosa cells was assessed by FACS; the lower-right quadrant represents the early phase apoptosis rate. FACS, fluorescence activated cell sorting; FITC, fluorescein isothiocyanate.

Article Snippet: ZM447439 (Axon Medchem LLC, Reston, VA, USA), an inhibitor of Aurora B, was dissolved in DMSO at concentrations of 10, 50, and 100 mM, and stored at −20°C.

Techniques: Cell Culture, Standard Deviation, Fluorescence, FACS

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: (A) Plot of cell viability against concentration of ZM447439 for both CEM/AKB4 and parental CEM cells as determined by cytotoxicity assay. (B) of the same experiment performed in the presence of the P-glycoprotein inhibitor verapamil. Points are the means, and bars are the SEM of at least three independent experiments.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques: Concentration Assay, Cytotoxicity Assay

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: Relative resistance of CEM/AKB4 cells to cytotoxic agents compared to parental CCRF-CEM cells.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques:

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: (A) AurkB gene expression as determined by real-time PCR. Expression is displayed as relative ΔΔCt values of CEM/AKB4 compared to that for CEM with Ct values normalised to the cyclophilin-A gene (PPIA). (B) Aurora B protein expression determined by western blot. The densitometric volume of the Aurora B band is expressed relative to the densitometric volume of the loading control gene GAPDH. Error bars represent the SEM of three independent experiments. * p<0.05, ** p<0.005. (C) Detection of phospho Histone H3(Ser10) in CEM and CEM/AKB4 cells treated for 24 hr with increasing concentrations of ZM447439 by western blotting. Shown are representative blots from three independent experiments.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: Mitotic index of CEM and CEM/AKB4 cells in the presence and absence of 4 µM ZM447439.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques:

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: Docked poses were compared between wild-type and mutant Aurora B for (A, B) ZM447439, (C, D) hesperadin and (E, F) aminothiazole inhibitor. Hydrogen bonds referred to in the text highlighted in yellow.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: Cells were grown either in vehicle alone or in 4 µM ZM447439 and proliferation was determined at indicated timepoints as the corrected absorbance using the Alamar blue assay measured spectrophotometrically. Error bars represent the SEM of three independent experiments.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques: Alamar Blue Assay

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: A) CEM/AKBMDR1 mRNA expression in ZM447439 resistant CEM cells as determined by real-time PCR. Expression is displayed as relative ΔΔCt values of CEM, CEM/AKB4, CEM/AKB4 and CEM/AKB4 cells compared to that for CEM/VCRR cells with Ct values normalised to the cyclophilin-A gene (PPIA). Error bars represent the SEM of three independent experiments. B) Cytotoxicity assays of doxorubicin against CEM and CEM/AKB4, 8, and 16 cells.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: A) Levels of cleaved PARP in cells treated with indicated concentrations of ZM447439 for 24 h as determined by western blot. B) Proportion of apoptotic cells in both untreated and CEM and CEM/AKB cells treated with 16 µM ZM447439 for 24 h.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Evolution of Resistance to Aurora Kinase B Inhibitors in Leukaemia Cells

doi: 10.1371/journal.pone.0030734

Figure Lengend Snippet: Levels of phosphorylated Histone H3 in CEM and CEM/AKB cells in the presence or absence of 16 µM ZM447439.

Article Snippet: ZM447439 , CEM , 3.40±0.37×10 −7 , - , .

Techniques:

Journal: Cell Cycle

Article Title: A role for p38 in transcriptional elongation of p21 CIP1 in response to Aurora B inhibition

doi: 10.4161/cc.25100

Figure Lengend Snippet: Figure 1. Activation of p53 and p21 in response to Aurora B inhibition. (A) U2OS cells were treated with 1 µM ZM447439 for 24 h. Nuclei were stained with Hoechst 33258. p21 was detected by immunostaining. Bar: 50 µm. (B) U2OS cells treated with 1 µM ZM447439 for 24 and 48 h were analyzed by FACS. (C) Cells were treated with 1µM ZM447439 and levels of p53 and p21 were determined by immunoblotting. β-actin served as a control for equal loading (D) U2OS cells were treated with 1 µM ZM447439 for 72 h. Four days later, senescent cells were detected by staining for β-galactosidase. (E) Cells stably expressing a control shRNA or a p53-specific shRNA were treated with ZM447439 for 36 h and p21 protein levels were determined by immunoblotting (F) U2OS cells were treated with AZD1152 as indicated and p21 levels were determined by immunoblotting. β-actin served as a control for equal loading. (G) U2OS cells were transfected with a control siRNA (ctrl) or with an Aurora B-specific siRNA. 48 h later, p21 levels were determined by immunoblotting. Tubulin served as control.

Article Snippet: Cells were treated with the indicated concentrations of ZM447439 (Enzo), AZD1152 (Selleckchem), SB202190 (Sigma), BIRB796 (Selleckchem), doxyrubicin (Sigma). siRNA oligonucleotides (MWG) were transfected using Lipofectamine RNAi Max (Invitrogen).

Techniques: Activation Assay, Inhibition, Staining, Immunostaining, Western Blot, Stable Transfection, Expressing, shRNA, Transfection

Journal: Cell Cycle

Article Title: A role for p38 in transcriptional elongation of p21 CIP1 in response to Aurora B inhibition

doi: 10.4161/cc.25100

Figure Lengend Snippet: Figure 2. Polyploidization in response to Aurora B inhibition is increased in the absence of p21. (A) HCT116 cells were treated with ZM447439 or AZD1152 for 24 h or 48 h. p21 levels were determined by immunoblotting. β-actin served as a control. (B) FACS assays of HCT116 cells treated either with 0.5 µM and 1 µM ZM447439 or with 50 nM and 100 nM AZD1152 for 24 h or 48 h. (C) HCT116 wild type and p21−/− cells were treated with 1 µM ZM447439 for 24 or 48 h. Cell cycle distribution was analyzed by FACS.

Article Snippet: Cells were treated with the indicated concentrations of ZM447439 (Enzo), AZD1152 (Selleckchem), SB202190 (Sigma), BIRB796 (Selleckchem), doxyrubicin (Sigma). siRNA oligonucleotides (MWG) were transfected using Lipofectamine RNAi Max (Invitrogen).

Techniques: Inhibition, Western Blot

Journal: Cell Cycle

Article Title: A role for p38 in transcriptional elongation of p21 CIP1 in response to Aurora B inhibition

doi: 10.4161/cc.25100

Figure Lengend Snippet: Figure 3. p38 is required for induction of p21 in response to Aurora B inhibition. (A) U2OS cells were treated for the indicated time with 1 µM ZM447439. Levels of p53, p21, phosphorylated p38 (p-p38) and total p38 were determined by immunoblotting. (B) Cells were pretreated with DMSO or 10 µM SB202190 for 2 h and then treated with 1 µM ZM447439 for 24 h. Levels of p21 and p53 were determined by immunoblotting. (C) Cells were pretreated with DMSO or 1 µM BIRB796 for 2 h and then treated with 50 nM AZD1152 for 24 h. Levels of p21 and p53 were determined by immunoblotting. (D) HCT116 cells were pretreated with the p38 MAP kinase inhibitor BIRB796 (1 µM) for 2 h and then treated with ZM447439 (1 µM) for 24 h. p21 and p53 protein levels were determined by immunoblotting. (E) HCT116 cells were treated as in (D), but with a different Aurora B inhibitor, AZD1152 (50 nM). (F) Cells were pre-treated with 10 µM SB202190 for 2 h, as indicated. Cells were than treated either with DMSO, 1 µM ZM447439 or 1 µM doxorubicin (Dox). Levels of p21 were determined by immunoblotting.

Article Snippet: Cells were treated with the indicated concentrations of ZM447439 (Enzo), AZD1152 (Selleckchem), SB202190 (Sigma), BIRB796 (Selleckchem), doxyrubicin (Sigma). siRNA oligonucleotides (MWG) were transfected using Lipofectamine RNAi Max (Invitrogen).

Techniques: Inhibition, Western Blot

Journal: Cell Cycle

Article Title: A role for p38 in transcriptional elongation of p21 CIP1 in response to Aurora B inhibition

doi: 10.4161/cc.25100

Figure Lengend Snippet: Figure 4. p38 is required to prevent entry into S-phase when Aurora B is inhibited (A) Cells were pretreated with DMSO or 10 µM SB202190 for 2 h followed by treatment with 0.5 µM ZM447439 for 3 d. Before analysis, cells were pulse labeled with 15 µg/ml BrdU for 2 h. The percentage of BrdU-positive cells was determined by immunofluorescence. Error bars represent standard deviation of three independent experiments. At least 800 cells were counted. (B) Cells were treated with 0.5 µM ZM447439 and 10 µM SB202190 as indicated. The percentage of cells in the different phases of the cell cycle and of polyploid cells (> 4N) was determined by FACS. (C) Cells were plated in 10 cm dishes, pretreated with DMSO or 10 µM SB202190 for 2 h followed by treatment with 0.5 µM ZM447439 for 3 d. Ten days later, colonies were fixed and stained with crystal violet. (D) Cells were treated with 0.5 µM ZM447439 and 10 µM SB202190 for 6 h, as indicated. Apoptotic cells were determined by Annexin V staining. Four independent experiments were performed. The differences between ZM447439- and ZM447439 + SB202190-treated cells were statistically significant (p < 0.0253; Student’s t-test).

Article Snippet: Cells were treated with the indicated concentrations of ZM447439 (Enzo), AZD1152 (Selleckchem), SB202190 (Sigma), BIRB796 (Selleckchem), doxyrubicin (Sigma). siRNA oligonucleotides (MWG) were transfected using Lipofectamine RNAi Max (Invitrogen).

Techniques: Labeling, Immunofluorescence, Standard Deviation, Staining

Journal: Cell Cycle

Article Title: A role for p38 in transcriptional elongation of p21 CIP1 in response to Aurora B inhibition

doi: 10.4161/cc.25100

Figure Lengend Snippet: Figure 5. p38 is required for transcription of p21 but not for nuclear localization of p53 or for p21 protein or mRNA stability (A) U2OS cells were treated with DMSO, 1 µM ZM447439 or 1µM ZM447439 and 10 µM SB202190 for 24 h and p53 localization was determined by immunostaining. Nuclei were counterstained with Hoechst 33258 Scale bar: 25 µM. (B) U2OS cells were treated with ZM447439 or with ZM447439 and SB202190 and then with cycloheximide (CHX) for the indicated time points to block protein synthesis. p21 levels were determined by immunoblotting. Tubulin served as a control for equal loading. (C) U2OS cells were pretreated with DMSO or 10 µM SB202190 for 2 h and then treated with 1 µM ZM447439 for 24 h. Levels of p21 mRNA were determined by RT-qPCR. (D) U2OS cells were treated with ZM447439 or with ZM447439 and SB202190 and then with actinomycin to block transcription. p21 mRNA levels were determined by RT-qPCR.

Article Snippet: Cells were treated with the indicated concentrations of ZM447439 (Enzo), AZD1152 (Selleckchem), SB202190 (Sigma), BIRB796 (Selleckchem), doxyrubicin (Sigma). siRNA oligonucleotides (MWG) were transfected using Lipofectamine RNAi Max (Invitrogen).

Techniques: Immunostaining, Blocking Assay, Western Blot, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: A role for p38 in transcriptional elongation of p21 CIP1 in response to Aurora B inhibition

doi: 10.4161/cc.25100

Figure Lengend Snippet: Figure 6. p38 is required for transcriptional elongation of p21 in response to Aurora B inhibition. (A) Schematic diagram of the p21 gene locus and position of the amplicons used for ChIP analysis and primary transcript analysis. (B) ChIP assays were performed with chromatin from U2OS cells treated with 1 µM ZM447439 and 10 µM SB202190 or with a combination of both drugs. Chromatin was precipitated either with nonspecific IgG or with antibodies directed at p53. ChIP enriched DNA was amplified by qPCR using the indicated amplicons. The GAPDH2 promoter was analyzed as a control (C) Cells were treated as described in C. ChIP assays were performed with antibodies directed at total RNA polymerase II. (D) Cells were treated as described in (C). ChIP assays were performed with antibodies directed at RNA polymerase II phosphorylated at serine 2. (E) U2OS cells were pretreated with DMSO or 10µM SB202190 for 2 h and then treated with 1 µM ZM447439 for 24 h. Total RNA was isolated and subjected to RT-qPCR using primers specific for different regions of the primary p21 transcript.

Article Snippet: Cells were treated with the indicated concentrations of ZM447439 (Enzo), AZD1152 (Selleckchem), SB202190 (Sigma), BIRB796 (Selleckchem), doxyrubicin (Sigma). siRNA oligonucleotides (MWG) were transfected using Lipofectamine RNAi Max (Invitrogen).

Techniques: Inhibition, Amplification, Isolation, Quantitative RT-PCR