Journal: Acta pharmacologica Sinica

Article Title: Avasimibe exerts anticancer effects on human glioblastoma cells via inducing cell apoptosis and cell cycle arrest.

doi: 10.1038/s41401-020-0404-8

Figure Lengend Snippet: Fig. 6 Avasimibe induced cell cycle arrest in the U251 and U87 cells. a Western blot analysis showed that avasimibe induced cell cycle arrest in the G0/G1 phase by regulating the p53/p21/p27 pathway. b Western blot analysis showed that avasimibe arrested the cell cycle in the G2/M phase by regulating the p53/GADD45A and Aurora A/PLK1 pathways. c Schematic model for the mechanism of avasimibe in cell cycle arrest

Article Snippet: The primary antibodies against cleaved caspase-9, cleaved PARP, p21, p27, CDK2, cyclin E1, CDK4, cyclin D, and PLK1 were purchased from Cell Signaling Technology; antibodies against Bax, p53, Aurora A, CDK1, cyclin B1, and GAPDH were purchased from Proteintech.

Techniques: Western Blot

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A. Representative images of tissue microarray IHC, (n = 206) stained with AURKA/DAB-brown, hematoxylin-nuclei/blue. Scale bar-300 μm. Insets-x250 enlarged areas. B, C Quantification N-AURKA positive(+) cells as in A, 3 randomly-assigned fields, n = 100 cells/field. B Pathological stages (Normal = 32, DCIS = 24, IDC = 72, MIDC-LN = 32, ILC = 24) and C receptor-based subtypes (Normal = 32, TNBC = 39, HER2+ = 32, ER+/PR+ = 30). D WB analysis of nuclear/cytoplasmic fractionations, as indicated. E Quantification of AURKA in cytoplasm/nucleus, percent-of-total, normalized to controls. F Schematic outline of cell line production. G, H Immunofluorescence and WB analysis of AURKA-sublines produced in F stained with AURKA (green), RFP(red), DAPI-nuclei/blue; clones indicated as c1/c2/c3. Scale bar-10 μm. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Microarray, Staining, Immunofluorescence, Produced, Clone Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A Experimental design of xenograft study, n = 3 mice/group. B Quantification of Fluorescent-IHC using phospho-AURKA-T288 antibody in tumors. n = 50 mitotic-cells/tumor. C Quantification of final tumor volume/mm3. D Representative H&E images of lung metastases and DAB-IHC images of liver metastases stained with anti-RFP-antibody. Metastases outlined by black line. Scale bar-50 μm. E, F Quantification of metastases: number and area of metastases normalized to total area. G Lung and liver metastasis penetrance in mice treated with vehicle or MLN8237. One-way ANOVA, ±S.E.M. Tukey’s test. H Model of Nuclear-AURKA-HIF1 mediated gene expression. Normoxia: HIF1A is hydroxylated/ubiquitinated by PHDs/VHL resulting in degradation. Hypoxia: low-oxygen stabilizes HIF1A leading to HIF1A/B dimerization/activation, transactivation of hypoxia-response genes. Normoxia+N-AURKA: HIF1A/B activity is increased leading to metastasis. MLN8237 inhibits AURKA activity decreasing metastasis.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Staining, Gene Expression, Activation Assay, Activity Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A WB analysis of HIFs in cells with indicated antibodies, DMSO-vehicle or DMOG for 7 h. B Quantification of WB results as in A, fold of change over DMSO-control. C, D Representative images of Immunoprecipitation/WB analysis with indicated antibodies, WCL-whole cell lysate. E Quantification of ChIP qPCR against selected promoter region, normalized to total input. F WB analysis of ChIP (before de-crosslinking). G Mass-spectrometry analysis of AURKA-IP complexes: Venn diagram displaying the numbers of proteins found in the NLS- or NES-AURKA complexes. The 850 proteins/yellow were further filtered, nuclear/blue. Pie-chart showing distribution of N-AURKA binding partners based on functional Panther Gene Ontology terms. Tables showing selected nuclear protein classifications for H RNA-binding and I DNA-binding from PANTHER Gene Ontology. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Control, Immunoprecipitation, ChIP-qPCR, Mass Spectrometry, Binding Assay, Functional Assay, RNA Binding Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A Heat-map of differentially expressed genes (DEGs, mean values) in cells. Gene expression (n = 3167) is normalized log2 counts/million. B Principal Component Analysis of RNA-Seq libraries as in A. C Volcano-plot analysis of RNAseq data as in A NLS-AURKA vs. control, log2 fold changes of gene expression on the x-axis, FDR statistical significance (−log10 p value) on the y-axis; upregulated (red), downregulated (black) genes in NLS, all genes (blue). Genes with at least a >1.5 fold change and FDR < 0.01 are displayed. D Visualization of Gene Ontology terms for NLS-AURKA vs. control; up/downregulated GOterms (FDR < 0.1) are depicted as circles; the distance indicates the relationship between terms: closer distance means higher similarity. Color and size of circles indicate significance of differential expression of an individual GO term in log10 p value. E WB analysis of selected RNA-seq target proteins as in A. F Quantification of WB in E, fold change over control. G GSEA comparison NLS-AURKA vs. control for selected GOterm gene sets. H Heat-map of HIF-dependent DEGs. I Predicted upstream regulators: activated (yellow), inhibited (blue) using IPA activation z-score. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Gene Expression, RNA Sequencing, Control, Quantitative Proteomics, Comparison, Activation Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A WB analysis of HIFs in cells as indicated, treated with siRNA: control-scr or anti-HIF1A/B. B Quantification of WB results in A, fold of change over siScr. C Individual cell movement tracking-plots toward chemoattractant. Cell lines as indicated. Graphs of D total distance, E cell-body directionality, F cell speed. G qPCR analysis of select genes in control and NLS-AURKA cells with siScr and siHIF1A/B, fold change over control-siScr. H qPCR analysis of FOXM1 in control and NLS-AURKA cells treated with siScr and siHIF1A/B, fold change over control-siScr. I, J qPCR analysis of FOXM1, HIF1A, and HIF1B in control and NLS-AURKA cells treated with siScr and siFOXM1, fold change over control-siScr. HIF1B: (unpaired t test: con-Scr vs NLS-AURKA-Scr). One-way ANOVA, ±S.E.M. Tukey’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in oncology

Article Title: Aurora kinase A promotes hepatic stellate cell activation and liver fibrosis through the Wnt/β-catenin pathway.

doi: 10.3389/fonc.2024.1517226

Figure Lengend Snippet: FIGURE 1 Upregulated AURKA in liver fibrosis is associated with HSC activation. (A) Relative AURKA mRNA expression in human liver fibrosis samples obtained from the GEO dataset (GSE84044). (B) A positive correlation was observed between the mRNA levels of AURKA and those of ACTA2 and COL1A1 in human fibrotic liver samples from the GEO dataset (GSE38941). (C) Representative images of AURKA immunohistochemistry staining in human liver fibrosis/cirrhosis (n =9) and benign hepatic diseases (n =3) and the results of semi-quantitative immunohistochemical analysis. Scale bars, 25mm. (D) Relative mRNA expression of AURKA in CCl4 (n =5) and Bile duct ligation (n =7) induced liver fibrosis mouse model. (E) Representative images of a- SMA and AURKA staining in liver sections of mice treated with CCl4 and control group for 4 or 8 weeks. Scale bars, 100mm. (F) Represent double immunofluorescence images of a-SMA (red) and AURKA (green) in fibrotic liver tiusse. Scale bars, 25mm. (G) Double-immunofluorescence staining of AURKA and a-SMA in mouse primary HSCs cultured for 0 and 5 days in vitro. Scale bars, 100mm. Data presented as means ± SD. NS, not significant; *P < 0.05; **P < 0.01.

Article Snippet: In the dual immunofluorescence staining procedure, cells were fixed using ice-cold methanol and co-stained with AURKA (1/100; frontiersin.org Biorbyt; orb224015) and a-SMA (1/500; Abcam; ab7817).

Techniques: Activation Assay, Expressing, Immunohistochemistry, Staining, Immunohistochemical staining, Ligation, Control, Cell Culture, In Vitro