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: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a The Claudin low (CL)-TNBC subgroup (72 cases) showed an average age at diagnosis of 61.9 years, while the overall survival average was 126.5 months. The median months' survival calculated for the whole follow-up was 27.96 months. In total, 12/72 cases (17%) exhibited AURKA alterations (mRNA up-regulation and/or copy number variations), while none of 72 patients harbored deletions or down-regulations of AURKA. Survival analysis showed that aberrant AURKA expression was significantly associated with reduced patient overall survival ( p -value = 0.00584). b Immunoblot assay showing expression of total and phosphorylated AURKA in BT-549 cells before and after treatment with TGF-β1 (10 ng/ml) for 48 h. c Densitometry analysis showing the fold change of total and phosphorylated AURKA protein levels in BT-549 TNBC cells normalized to α-Tubulin. Experiments were performed in triplicate. d BT-549 cells were treated with TGF-β1 (10 ng/ml) for 48 h. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as control. Graph showing the average of ALDH1 High cells from three independent experiments (±s.d.). e Immunoblot assay showing expression of total AURKA in BT-549 cells infected with scrambled and AURKA lenti-shRNAs for 48 h. Densitometry analysis showing the fold change of total AURKA protein levels in BT-549 TNBC cells normalized to α-Tubulin. f BT-549 cells were treated with 10 ng/ml TGF-β1 and lenti-shRNAs. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. Graph showing the average of ALDH1 High cells from three independent experiments (±s.d.). g BT-549 cells were treated with 10 ng/ml TGF-β1, scrambled lenti-shRNAs, and lenti-shRNAs targeting SMAD3. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. Graph showing the average of ALDH1 High cells from three independent experiments (±s.d.).

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: Expressing, Western Blot, Incubation, Activity Assay, Infection

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a Immunoblot assay showing expression of TGβR1, TGβR2, SMAD3 (total and phosphorylated), and AURKA (total and phosphorylated) proteins in MDA-MB 231 and SUM149-PT cells. Densitometry analysis showing the fold change of protein levels in MDA-MB 231 and SUM149-PT cells normalized to α-Tubulin. b SUM149-PT cells were treated with 10 ng/ml TGF-β1, lenti-shRNAs, or alisertib. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. c Graph showing the average of ALDH1 high cells from three independent experiments (±s.d.). d SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 1000 SUM149-PT cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-ShRNAs and were incubated for 8 days to monitor MPS growth. Graph showing the average of ALDH1 High cells from three independent experiments (±s.d.). e FACS sorting analysis was performed on SUM149-PT secondary MPS to separate ALDH1 high from ALDH1 low cells. 10,000 ALDH1 high and ALDH1 low cells were then cultured for 8 days under non-adherent conditions to form tertiary MPS. Cancer cells were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and the MPS area was measured using the NIH Image-J software. Graph showing the average of ALDH1 High and ALDH1 low MPS area from three independent experiments (+/− s.d.). f GFP-tagged SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 10,000 SUM149-PT cells derived from tertiary MPS were then treated with DMSO (control) or 1 μM A37 (ALDH inhibitor) for 8 days. MPS area was measured using the NIH Image-J software. Graph showing the average of MPS area from three independent experiments (±s.d.).

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Incubation, Activity Assay, Cell Culture, Derivative Assay, Infection, Labeling, Software

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a BT-549 cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 10,000 cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-shRNAs, treated with 10 ng/ml TGF-β1 in the presence of DMSO (control) or 10 nM docetaxel (DTX) and incubated for 8 days to monitor MPS growth. MPS were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and the MPS area was measured using the NIH Image-J software. b Graph showing the average of MPS area from three independent experiments (±s.d.). c GFP-tagged SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-shRNAs and treated with DMSO (control) or 10 nM DTX for 8 days to monitor MPS growth. MPS area was measured using the NIH Image-J software. d Graph showing the average of MPS area from three independent experiments (±s.d.).

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: Cell Culture, Derivative Assay, Infection, Incubation, Labeling, Software

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a RNA-Seq analysis was performed on MDA-MB 231 cells treated with 10 ng/ml TGF-β1 for 48 h. 14,239 genes were differentially expressed between control and TGF-β1 groups. b STRINGdb software was used to identify a SNAI1/MMP9/FN1 Network. c Quantification analysis showing FN1, MMP9, and SNAI1 expression before and after TGF-β1 treatment. Experiments were performed in triplicate with a p -value < 0.05. d Real-time quantitative RT-PCR to detect SNA1, FN1, and MMP9 gene expression using MDA-MB 231 cells treated with 10 ng/ml TGF-β1 and infected with scrambled Lenti-shRNA or Lenti-shRNA targeting AURKA ( Origene ) for 48 h. Three independent experiments were performed in triplicate (±S.D. and P- value < 0.05).

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Software, Expressing, Quantitative RT-PCR, Infection, shRNA

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a BT-549 cells were treated with 10 ng/ml TGF-β1, scrambled lenti-shRNAs and lenti-shRNAs targeting SNAI1. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. Graph showing the average of ALDH1 High cells from three independent experiments (±s.d.). b SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then infected with scrambled or SNAI1 lenti-shRNAs and treated with DMSO (control) or 10 nM DTX for 8 days to monitor MPS growth. MPS were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and MPS area was measured using the NIH Image-J software. c Graph showing the average of MPS area from three independent experiments (±s.d.). d In total, 12,000 cells infected with scrambled or AURKA and/or SNAI1 lenti-shRNAs were plated on 96-well costar plates and Real-time wound healing assay was performed by using the IncuCyte Instrument. Experiments were performed in triplicate (±s.d.). e In total, 50,000 TNBC-M40 cells infected with Luciferase lenti-vectors and scrambled Lenti-shRNA or SNAI1 scrambled Lenti-shRNA constructs were transplanted into the mammary fat pad of female NSG mice (three animals per group). Tumorigenic capacity was monitored in living animals at 0 (2 h post-injection was used as the control for cell viability) and 7 days post-injection by luciferase imaging. f The tumor Growth Area was quantified using ImageJ-NIH Software and represents the average of three animals per group.

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: Incubation, Activity Assay, Cell Culture, Derivative Assay, Infection, Labeling, Software, Wound Healing Assay, Luciferase, shRNA, Construct, Injection, Imaging

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a Immunoblot analysis showing higher phospho-SMAD3 and phospho-AURKA in TNBC-M14 and TNBC-M25 tertiary MPS compared to MDA-MB 231 used as control. Graphs showing the densitometric quantification of total and phosphoproteins expression normalized to Tubulin. b GFP-tagged TNBC-M14 and TNBC-M25 cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then treated with DMSO (control) or DTX for 8 days to monitor MPS growth. MPS area was measured using the NIH Image-J software. c Graph showing the average of MPS area from three independent experiments (±s.d.). d TNBC-M14 and TNBC-M25 cells were treated with 50 nM galunisertib, 50 nM alisertib, and combination for 48 h. Expression and cellular localization of Vimentin (green) was assessed by Immunofluorescence employing the Zeiss Confocal Fluorescent Microscope . Experiments were performed in triplicate with similar results. e Adherent and mammospheres-derived TNBC-M14 and TNBC-M25 were treated with 50 nM galunisertib (Gala), 50 nM alisertib (Alis), and combination. After 48 h incubation, ALDH activity was measured by FACS analysis on 10,000 events. ALDH inhibitor DEAB was used as control. Experiments were performed in triplicate (±s.d.). f TNBC-M14 and TNBC-M25 cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then treated with 50 nM galunisertib, 50 nM alisertib, and combination for 8 days. MPS were labeled in red using the CellTracker Red CMTPX Dye . g MPS area was measured using the NIH Image-J software. Experiments were performed in triplicate (±S.D. and P -value < 0.005).

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: Western Blot, Expressing, Cell Culture, Derivative Assay, Software, Immunofluorescence, Microscopy, Incubation, Activity Assay, Labeling

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: a , b Real-time apoptosis assay of TNBC-M14 and TNBC-M25 3D-MPS treated with 10 nM Docetaxel, 50 nM Galunisertib, and 50 nM Alisertib as single agents and in combination. Apoptotic cells were stained in red with ANNEXIN-V and quantified using the Cell Player System (IncuCyte, BioEssen). Experiments were performed in triplicate (±S.D. and P- value < 0.05). c Representative images of TNBC-M14 and TNBC-M25 3D-MPS treated with 10 nM Docetaxel, 50 nM Galunisertib, and 50 nM Alisertib as single agents and in combination. Apoptotic cells were stained in Red with ANNEXIN-V and quantified in real-time using the Cell Player System (IncuCyte, BioEssen). d TGF-β/AURKA oncogenic axis promotes the enrichment of chemoresistant ALDH high BTICs: Bulk TNBC cells show a chemosensitive ALDH low phenotype. Aberrant activation of TGF-β/AURKA/SNAI1 oncogenic axis induces TNBC plasticity resulting in the enrichment of ALDH1 high BTICs with intrinsic resistance to standard chemotherapeutic agents. Dual pharmacologic inhibition of TGF-β and AURKA pathways will impair TNBC plasticity and restore chemosensitivity through the selective targeting of ALDH1 high BTICs.

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lenti-vectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: Apoptosis Assay, Staining, Activation Assay, Inhibition

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: Functional roles of 12 hub genes.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques: Functional Assay, DNA Synthesis, Activation Assay

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: Survival analysis of the 12 hub genes in MED based on the GSE30074 database. (A) AURKA, (B) BUB1B, (C) CCNB1, (D) CCNB2, (E) CHEK1, (F) KIF11, (G) KIF20A, (H) MAD2L1, (I) MCM6, (J) NCAPG, (K) RFC4, (L) RRM2; P < 0.05 was considered statistically significant.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques:

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: Survival analysis of AURKA and KIF20A based on the GSE85217 database. (A) AURKA, (B) KIF20A. P < 0.05 was considered statistically significant.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques:

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: The different expressions of AURKA and KIF20A in the three databases. (A) Expressions of AURKA in GSE39182, (B) Expressions of AURKA in GSE74195, (C) Expressions of AURKA in GSE86574, (D) Expressions of KIF20A in GSE39182, (E) Expressions of KIF20A in GSE74195, (F) Expressions of KIF20A in GSE86574.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques:

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: AURKA’s biological involvement in tumors. (A) AURKA expression in different tumors. (B) AURKA gene STRING interaction network preview of interacting proteins (showing top 20 STRING interactants). (C) Illustration of KEGG pathway analysis findings using the ClueGO plugin in Cytoscape. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: Pathway and process enrichment analysis of AURKA and top 20 interacting proteins.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques:

Journal: Frontiers in Oncology

Article Title: Integrated Analysis of Transcriptome Data Revealed AURKA and KIF20A as Critical Genes in Medulloblastoma Progression

doi: 10.3389/fonc.2022.875521

Figure Lengend Snippet: Immunohistochemistry analysis of AURKA and KIF20A expression in Medulloblastoma tissues from Tianjin Huanhu Hospital (normal brain, n = 4; MED, n = 10). Scale bar = 50μm. Below is a list of statistical quantitative analyses. Data are mean ± SD. ***P < 0.001, one-way ANOVA.

Article Snippet: AURKA mouse monoclonal antibody (1:400, Proteintech, 66757-1-IG) and KIF20A rabbit polyclonal antibody (1:400, Proteintech, 15910-1-AP) were stained overnight at 4°C, and the second antibody was subjected to incubation for 1 hour at ambient temperature.

Techniques: Immunohistochemistry, Expressing