Journal: eLife

Article Title: Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis

doi: 10.7554/eLife.11402

Figure Lengend Snippet: ( A ) In vitro kinase activity of Aurora-A,-B,-C for H3 T118 peptide. ( B ) Immunofluorescence of pro-metaphase HeLa cells cotransfected with H2B:RFP and siRNA to Aurora-A (bottom) or control scrambled siRNA (top). Scale bar = 5 μm. ( C ) Cytokinesis in 293TR cells transiently transfected with H3-YFP plasmids. YFP (yellow) and DNA stained with DAPI (blue). Scale bar = 5 μm. ( D ) Quantitation of C (n=30 cells in anaphase, **p=0.01, by Fishers exact test). Error bars represent SD of the mean (SDM). ( E ) Quantitative data of live cell imaging showing differences in average length in cytokinesis during live cell imaging (n = 50 cells, **p<0.01 and ***p<0.001 by unpaired student t-test). ( F ) Error correction assay for 293TR stable cell lines expressing H3. Inhibition of Aurora-B with ZM447439 represents an extreme case of inability to correct error. Scale bar =10 μm ( G ) Quantitation of cells with misaligned chromosomes on the metaphase plate as in F. (*p<0.05 and ***p<0.001 by Fishers exact test). Error bars represent SDM. DOI: http://dx.doi.org/10.7554/eLife.11402.006

Article Snippet: All inhibitors were used at the listed concentrations MG132 (20 μM in ETOH), RO-3306 (9 μM, Enzo Life Sciences, ALX-270-463-M001, in DMSO), Nocodazole (100 mg/ml, Sigma, M1404, in ETOH), Colcemid (0.01 μg/mL, Roche 10295892001), PLK-1 inhibitor BI-2536 (100 nM, Selleck chemicals, Houston, TX USA, S1109, in DMSO), Caffeine (80 nM, Sigma C0750, in DMEM), Aurora-B inhibitor ZM447439 (2 μM, Tocris Biosciences, S1103, in DMSO), Calyculin A (50 nM, Tocris Biosciences, in EtOH), Aurora-B inhibitor Hesperidin (1 μM Selleck chemicals S2309, in DMSO), Aurora-A inhibitor VX-680 (1 μM, Selleck chemicals, S1048, in DMSO), Aurora-A inhibitor MLN 8237 (1 μM, Selleck Chem, S1133, in DMSO), Topoisomerase II inhibitor ICRF 193 (10 μM, Sigma, U4659, in DMSO).

Techniques: In Vitro, Activity Assay, Immunofluorescence, Control, Transfection, Staining, Quantitation Assay, Live Cell Imaging, Stable Transfection, Expressing, Inhibition

Journal: Cell Chemical Biology

Article Title: Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G 0 /G 1 Arrest by Inhibiting Deubiquitinase USP2a

doi: 10.1016/j.chembiol.2017.03.002

Figure Lengend Snippet: Impact of LCAHA on the Expression and Stability of Cyclin D1 (A) The expression of cyclin D1-encoding mRNA ( CCND1 ) was evaluated by RT-PCR. The cells were treated with DMSO or 10 μM LCAHA for 48 hr. (B and C) The cells were treated with DMSO or LCAHA for 48 hr, and cycloheximide (CHX) was added for the last 15–60 min, followed by western blot analysis of cyclin D1 expression. The graph in (C) presents densitometry analysis of the expression level of cyclin D1 (B) and shows mean ± SEM values from three independent experiments. The statistical significance was evaluated using a t test on mean t 1/2 values from these three experiments. (D and E) Western blot analysis of the expression of cyclin D1, phospho-Akt, and phospho-GSK-3β in HCT116 p53 wt cells after the treatment with 5 μM LCAHA for the indicated time periods. (D) Western blot images from the representative experiment. (E) Densitometry analysis of the expression level of cyclin D1, P-Akt(S473), and P-GSK-3β(S9) normalized to α-tubulin. The graphs show mean ± SD from three independent experiments. Statistical significance was evaluated using a t test: *p < 0.05, **p < 0.01. (F) HCT116 cells were treated with DMSO (marked with D), or 5 or 20 μM LCAHA for 48 hr, followed by western blot analysis of the expression of Aurora A and cyclin A1. The graphs present densitometry analysis of the proteins' expression and show mean ± SEM from three independent experiments. Statistical significance was evaluated using one-way ANOVA with Tukey's post hoc test: *p < 0.05, **p < 0.01.

Article Snippet: The following antibodies and dilutions were used: rabbit polyclonal anti-cyclin D1 (1:200, Santa Cruz Biotechnology (SCBt), cat. sc-753), mouse monoclonal anti-Cyclin D3 (1:1 000, Cell Signaling Technology (CST), cat. 2936), polyclonal anti-p53 (1:200, SCBt, cat. sc-6243), rabbit monoclonal anti-p21 (1:1 000, CST, cat. 2947), rabbit monoclonal anti-p27 (1:1 000, CST, cat. 3686), mouse monoclonal anti-Cyclin A2 (1:1 000, CST, cat. 4656), mouse monoclonal anti-Cyclin E1 (1:1 000, CST, cat. 4129), rabbit monoclonal anti-p-Akt(S473) (1:1 000, CST, cat. 4060), rabbit monoclonal anti-p-GSK-3β(S9) (1:1 000, CST, cat. 5558), mouse monoclonal anti-Aurora A (1:100, SCBt, cat. sc-373856), rabbit polyclonal anti-cyclin A1 (1:200, SCBt, cat. sc-7252), rabbit monoclonal anti-GAPDH (1:4 000, CST, cat. 2118), rabbit monoclonal anti-α-Tubulin (1:4 000, CST, cat. 2125), goat peroxidase-conjugated anti-rabbit (1:2 000, CST, cat. 7074), horse peroxidase-conjugated anti-mouse (1:2 000, Cell Signaling, cat. 7076).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cell Chemical Biology

Article Title: Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G 0 /G 1 Arrest by Inhibiting Deubiquitinase USP2a

doi: 10.1016/j.chembiol.2017.03.002

Figure Lengend Snippet:

Article Snippet: The following antibodies and dilutions were used: rabbit polyclonal anti-cyclin D1 (1:200, Santa Cruz Biotechnology (SCBt), cat. sc-753), mouse monoclonal anti-Cyclin D3 (1:1 000, Cell Signaling Technology (CST), cat. 2936), polyclonal anti-p53 (1:200, SCBt, cat. sc-6243), rabbit monoclonal anti-p21 (1:1 000, CST, cat. 2947), rabbit monoclonal anti-p27 (1:1 000, CST, cat. 3686), mouse monoclonal anti-Cyclin A2 (1:1 000, CST, cat. 4656), mouse monoclonal anti-Cyclin E1 (1:1 000, CST, cat. 4129), rabbit monoclonal anti-p-Akt(S473) (1:1 000, CST, cat. 4060), rabbit monoclonal anti-p-GSK-3β(S9) (1:1 000, CST, cat. 5558), mouse monoclonal anti-Aurora A (1:100, SCBt, cat. sc-373856), rabbit polyclonal anti-cyclin A1 (1:200, SCBt, cat. sc-7252), rabbit monoclonal anti-GAPDH (1:4 000, CST, cat. 2118), rabbit monoclonal anti-α-Tubulin (1:4 000, CST, cat. 2125), goat peroxidase-conjugated anti-rabbit (1:2 000, CST, cat. 7074), horse peroxidase-conjugated anti-mouse (1:2 000, Cell Signaling, cat. 7076).

Techniques: Recombinant, Protease Inhibitor, Isolation, Western Blot, Staining, Plasmid Preparation, High Throughput Screening Assay, Activity Assay, Integrity Assay, Caspase-Glo Assay, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Biochimica et biophysica acta

Article Title: Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

doi: 10.1016/j.bbamcr.2013.05.016

Figure Lengend Snippet: Fig. 4. Effect of aurora A kinase inhibitor MLN8237, aurora B kinase inhibitor AZD1152-HQPA, and Cdk1 inhibitor RO3306 on 17α-E2-induced G2/M-arrest (A), Δψm loss (B), and Bak activation (C) in JT/Neo cells. After treatment with 10 μM 17α-E2 in the absence or in the presence of 12.5 nM MLN8237, 12.5 nM AZD1152-HQPA or 6 μM RO3306 for 20 h, the cells were harvested. Flow cytometric analyses of cell cycle distribution, Δψm loss, and Bak activation were performed as described in Section 2. A representative study is shown; two additional experiments yielded similar results.

Article Snippet: The Cdk1 inhibitor RO3306 was purchased from Tocris Bioscience (Ellisville, MO, USA), and the aurora A kinase inhibitor MLN8237 and aurora B kinase inhibitor AZD1152-HQPA were obtained from Selleck (Huston, TX, USA).

Techniques: Activation Assay

Journal: Biochimica et biophysica acta

Article Title: Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

doi: 10.1016/j.bbamcr.2013.05.016

Figure Lengend Snippet: Fig. 5. Western blot analyses of phosphorylated Cdk1 (Thr-161 and Tyr-15), Cdk1, cyclin B1, phosphorylated histone H1, histone H1, phosphorylated Cdc25C (Thr-48), Cdc25C, phosphorylated aurora A (Thr-288), aurora A, phosphorylated histone H3 (Ser-10), histone H3, phosphorylated Bcl-2 (Thr-56 and Ser-70), Bcl-2, phosphorylated Mcl-1 (Ser-159/Thr-163), Mcl-1, electrophoretic mobility retardation of Bim isoforms, caspase-3, and α-tubulin in JT/Neo cells after treatment with 17α-E2 in the absence or presence of MLN8237, AZD1152-HQPA, or RO3306. After individual cell lysates were prepared, Western blot analyses were performed as described in Section 2. Symbol: ←*, phosphorylated form of Cdc25C. A representative study is shown; two additional experiments yielded similar results.

Article Snippet: The Cdk1 inhibitor RO3306 was purchased from Tocris Bioscience (Ellisville, MO, USA), and the aurora A kinase inhibitor MLN8237 and aurora B kinase inhibitor AZD1152-HQPA were obtained from Selleck (Huston, TX, USA).

Techniques: Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Activation of EIF4E by Aurora kinase A depicts a novel druggable axis in everolimus resistant cancer cells

doi: 10.1158/1078-0432.CCR-16-2141

Figure Lengend Snippet: Overexpression of AURKA using adenoviral particles in FLO-1 (A & B) and SK-GT-4 (C & D) cells increased p-EIF4E (S209) and c-MYC protein levels, and the activity of a dual Renilla-firefly-luciferase pcDNA3-rLuc-PolioIRES-fluc reporter that measures cap-dependent/independent translation. E) FLO-1 Parental, FLO-1 RAD-R (Pool), FLO-1 RAD-R (C1), and FLO-1 RAD-R (C2) were subjected to clonogenic cell survival assay in response to everolimus. IC50 of resistant cells is significantly higher than that of parental cells. G) Clonogenic cell survival of SK-GT-4 parental and RAD-R (Pool) cells in response to RAD001indicated a significant increase of IC50 of resistant cells relative to parental cells. Western blot analysis of FLO-1 parental, FLO-1 RAD-R (Pool), FLO-1 RAD-R (C1), FLO-1 RAD-R (C2) (F), SK-GT-4 (Parental), and SK-GT-4 RAD-R (Pool) cells (H) showed an increase in p-EIF4E (S209), p-AKT (S473), p-ERK1/2 (S217/221), and c-MYC protein levels in resistant cells relative to parental cells.

Article Snippet: AKT inhibitor, MK2206, MEK inhibitor, trametinib, AURKA inhibitor, alisertib, and mTOR inhibitor, RAD001 (everolimus) were purchased from Selleck chemicals (Houston, TX). p-AURKA (T288), AURKA, p-EIF4E (S209), EIF4E, p-mTOR (S2448), mTOR, p-4EB-P1 (S65), 4EB-P1, p-AKT (S473), AKT, MNK, p-MNK (T197/202), and PP2A antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Over Expression, Activity Assay, Luciferase, Clonogenic Cell Survival Assay, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Activation of EIF4E by Aurora kinase A depicts a novel druggable axis in everolimus resistant cancer cells

doi: 10.1158/1078-0432.CCR-16-2141

Figure Lengend Snippet: A) FLO-1 RAD-R (C1) and SK-GT-4 RAD-R (Pool) resistant cells were treated with AKT inhibitor (MK2206) for 6 h, and subjected to Western blot analysis. B) The same cells were treated with MEK1/2 inhibitor (Tramitinib) for 6 h and analyzed by immunoblotting. Data showed that inhibition of AKT or MAPK/ERK signaling pathways had no effect on p-EIF4E (S209) and c-MYC protein levels. C) Knockdown of AURKA in RAD001 resistant FLO-1or SK-GT-4 cells downregulated p-EIF4E (S209) and c-MYC proteins, with or without RAD001 treatment. D) Pharmacological inhibition of AURKA using alisertib led to downregulation of p-EIF4E (S209) and c-MYC proteins in FLO-1 and SK-GT-4 resistant cells, with or without RAD001 treatment.

Article Snippet: AKT inhibitor, MK2206, MEK inhibitor, trametinib, AURKA inhibitor, alisertib, and mTOR inhibitor, RAD001 (everolimus) were purchased from Selleck chemicals (Houston, TX). p-AURKA (T288), AURKA, p-EIF4E (S209), EIF4E, p-mTOR (S2448), mTOR, p-4EB-P1 (S65), 4EB-P1, p-AKT (S473), AKT, MNK, p-MNK (T197/202), and PP2A antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Inhibition, Protein-Protein interactions, Knockdown

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Activation of EIF4E by Aurora kinase A depicts a novel druggable axis in everolimus resistant cancer cells

doi: 10.1158/1078-0432.CCR-16-2141

Figure Lengend Snippet: AURKA inhibition by alisertib (A) or knockdown by siRNA (B) significantly downregulated cap-dependent translation in FLO-1 RAD-R (C1) and SK-GT-4 RAD-R (Pool) cells as compared to their respective parental cells. AURKA inhibition by alisertib (C) or knockdown by siRNA (D) significantly downregulated c-MYC transcriptional activity, as measured by 4xEMS luciferase reporter, in FLO-1 RAD-R (C1) and SK-GT-4 RAD-R (Pool) cells, relative to their respective parental cells. E) After knocking down of AURKA by siRNA, FLO-1 cells were subjected to translational chromatin immunoprecipitation (TrIP-Chip) assay and followed by qPCR analysis of total (input) and ploysomal RNA levels of AURKA, c-MYC, and CCND1. Data showed that knockdown of AURKA led to a significant decrease in the translated polysomal c-MYC and CCND1 RNA levels relative to their total RNA levels.

Article Snippet: AKT inhibitor, MK2206, MEK inhibitor, trametinib, AURKA inhibitor, alisertib, and mTOR inhibitor, RAD001 (everolimus) were purchased from Selleck chemicals (Houston, TX). p-AURKA (T288), AURKA, p-EIF4E (S209), EIF4E, p-mTOR (S2448), mTOR, p-4EB-P1 (S65), 4EB-P1, p-AKT (S473), AKT, MNK, p-MNK (T197/202), and PP2A antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Inhibition, Knockdown, Activity Assay, Luciferase, Chromatin Immunoprecipitation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Activation of EIF4E by Aurora kinase A depicts a novel druggable axis in everolimus resistant cancer cells

doi: 10.1158/1078-0432.CCR-16-2141

Figure Lengend Snippet: FLO-1 RAD-R (C1) cells (A) or SK-GT-4 RAD-R (Pool) cells (C) treated with alisertib or in combination with RAD001 were subjected to cell viability assay. Alisertib significantly inhibited the ability of RAD001 resistant cells to form colonies with or without RAD001 co-treatment. Parental FLO-1 cells (B) and SK-GT-4 cells (D) were treated with alisertib and subjected to cell viability assay. E) Human gastric MKN45 cell line displayed intrinsic resistance to RAD001 and sensitivity to alisertib as indicated by cell viability assay. Inhibition of AURKA with alisertib (F) or knockdown with siRNA (G) downregulated p-EIF4E (S209) and c-MYC protein levels as assayed by Western blotting. H) AURKA inhibition (left panel) or knockdown (right panel) significantly downregulated cap-dependent translation in MKN45 cells, as determined by a dual Renilla-firefly-luciferase pcDNA3-rLuc-PolioIRES-fluc reporter that measures cap-dependent/independent translation. I) AURKA inhibition (left panel) or knockdown (right panel) significantly downregulated c-MYC transcriptional activity in MKN45 cells, as measured by the 4xEMS luciferase reporter.

Article Snippet: AKT inhibitor, MK2206, MEK inhibitor, trametinib, AURKA inhibitor, alisertib, and mTOR inhibitor, RAD001 (everolimus) were purchased from Selleck chemicals (Houston, TX). p-AURKA (T288), AURKA, p-EIF4E (S209), EIF4E, p-mTOR (S2448), mTOR, p-4EB-P1 (S65), 4EB-P1, p-AKT (S473), AKT, MNK, p-MNK (T197/202), and PP2A antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Viability Assay, Inhibition, Knockdown, Western Blot, Luciferase, Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Activation of EIF4E by Aurora kinase A depicts a novel druggable axis in everolimus resistant cancer cells

doi: 10.1158/1078-0432.CCR-16-2141

Figure Lengend Snippet: A) Animals injected with MKN45 cells, and the tumors were allowed to grow until 200 mm3 in size, then treated with RAD001, alisertib or their combination for 5 weeks. Data indicated that alisertib alone or in combination with RAD001 significantly reduced tumor size in comparison with untreated or RAD001 alone treated groups. Although tumors in RAD001 treatment alone grew at a significantly slower rate than those in untreated group, they continued to grow, confirming the intrinsic resistant phenotype to RAD001. B and C) Immunohistochemistry analysis for cleaved caspase-3 expression, marker of apoptosis, and Ki-67 expression, marker of proliferation, in representative tumors of treated groups. D) A schematic diagram showing a proposed mechanism of RAD001 resistance. AURKA expression promotes RAD001 resistance through inhibition of PP2A, which leads to activation of EIF4E. Inhibition of AURKA by alisertib restores PP2A activity, thereby inhibiting EIF4E and inducing death in RAD001 resistant cancer cells.

Article Snippet: AKT inhibitor, MK2206, MEK inhibitor, trametinib, AURKA inhibitor, alisertib, and mTOR inhibitor, RAD001 (everolimus) were purchased from Selleck chemicals (Houston, TX). p-AURKA (T288), AURKA, p-EIF4E (S209), EIF4E, p-mTOR (S2448), mTOR, p-4EB-P1 (S65), 4EB-P1, p-AKT (S473), AKT, MNK, p-MNK (T197/202), and PP2A antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Injection, Comparison, Immunohistochemistry, Expressing, Marker, Inhibition, Activation Assay, Activity Assay