Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PDAC cells (n=3, *p < 0.05 versus control shRNA group). (C) Immunoprecipitation (IP) analysis of the levels of RIPK3 binding to RIPK1 and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (D) IP analysis of the levels of RIPK3 binding to RIPK1 and MLKL in control and stable AURKA-knockdown PANC1 and PANC2.03 cells. (E) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (F) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in HEK293 cells after expressed AURKA wild type and D274A mutant. (G) PANC1 cells were treated with CCT137690 (10 μM) or TNF (50 ng/ml) in the absence or presence of anti-TNFR1 antibody (1 mg/ml) for 24 hours and cell death was analyzed (n=3, *p < 0.05).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PANC1 and PANC2.03 cells (n=3, *p < 0.05 versus control shRNA group). (C, D) PANC1 and PANC2.03 cells were treated with XXVI (C) or AR-A014418 (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with XXVI or AR-A014418 for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F) Knockdown of RIPK3 and MLKL, but not RIPK1, inhibited XXVI- or AR-A014418-induced cell death (n=3, *p < 0.05 versus control shRNA group). (G–I) HEK293 cells were expressed with GSK3β WT and S9A mutant and then treated with CCT137690 (10 μM) for 24 hours. Protein level (G), cell viability (H), caspase-3 activity (H), and complex formation (I) were assayed.

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, shRNA, Mutagenesis, Activity Assay

Journal: Nature communications

Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells.

doi: 10.1038/s41467-018-05694-4

Figure Lengend Snippet: Fig. 4 Induction of multinucleation, G2/M arrest, and apoptosis in ARID1A−/−cells by AURKA inhibition. a Abnormal chromosomal segregation induced by AURKA silencing. ARID1A isogenic HCT116 cells were transfected with AURKA siRNA and analyzed for immunofluorescence of AURKA, α-tubulin, and nuclei/chromatin (HO33342). Scale bars, 10 µm. b Induction of multinucleation in ARID1A−/−cells by AURKA silencing. ARID1A isogenic HCT116 cells were transfected with AURKA siRNA and analyzed for immunofluorescence of α-tubulin (green) and nuclei/chromatin (blue). Images in the inlets (red square) are representative cells that were magnified and shown on the right side of each figure. Scale bars, 20 µm. c Percentage of multinuclear cells in ARID1A isogenic HCT116 cells treated with AURKA siRNA. Error bars represent s.d. **P < 0.01, Student’s t test. d Cell cycle analyses of ARID1A isogenic HCT116 cells treated with AURKAi. e Percentage of cell populations in each cell cycle phase. Error bars represent s.d. f, g Preferential induction of apoptosis in ARID1A−/−cells by AURKAi. Cells with or without AURKAi treatment were subjected to FITC–Annexin V apoptosis detection with a flow cytometer (f) and the percentage of apoptotic cells were quantitated (g). Error bars represent s.d. **P < 0.01, Student’s t test. h, i Preferential induction of apoptosis in ARID1A −/−cells by AURKA silencing (h) or AURKAi treatment (i). Active (cleaved) caspase-3 was used as a marker of apoptosis induction

Article Snippet: Each aliquot of protein sample was run on a SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Santa Cruz, sc-32761, 1:1000 dilution), CDC25C (Santa Cruz, sc-327, 1:2000 dilution), CDC2 (Santa Cruz, sc-54, 1:2000 dilution), GAPDH (Santa Cruz, sc-365062, 1:5000 dilution), HSP90 (Santa Cruz, sc-69703, 1:5000 dilution), αtubulin (Santa Cruz, sc-5286, 1:5000 dilution), p-CDC25C (Cell Signaling, #9529s, 1:1000 dilution), p-CDC2 (Cell Signaling, #9111s, 1:1000 dilution), AURKA (Cell Signaling, #14475s, 1:2000 dilution), and cleaved caspase 3 (Cell Signaling, #9661s, 1:2000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).

Techniques: Inhibition, Transfection, Cytometry, Marker

Journal: Nature communications

Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells.

doi: 10.1038/s41467-018-05694-4

Figure Lengend Snippet: Fig. 6 AURKA–CDC25C axis is a target for synthetic lethality in ARID1A-KO colorectal cancer cells. a Immunoblots showing upregulation of AURKA and phosphorylated CDC25C at Ser198 levels and downregulation of phosphorylated CDC2 at Tyr15 level in ARID1A−/−HCT116 cells. b, c Inhibition of CDC25C phosphorylation at Ser198 and increased phosphorylation of CDC2 at Tyr15 by AURKA silencing (b) and AURKAi treatment (c). d Immunofluorescence analysis of CDC25C localization in ARID1A isogenic cells treated with or without AURKA siRNA. Scale bars, 20 µm. e Synthetic lethality in ARID1A−/−HCT116 cells by CDC25 inhibitor II. f Synthetic lethality in ARID1A−/−HCT116 cells by PLK1 siRNA (siPLK1). Error bars represent s.d. *P < 0.05; **P < 0.01, Student’s t test. g Working model of the synthetic lethality between ARID1A and AURKA. In ARID1A wild-type (WT) cells, AURKA expression is negatively regulated by ARID1A, thereby reducing the activity of AURKA downstream pathway, including PLK1 and CDC25C. In ARID1A mutant (MT) cells, AURKA-PLK1-CDC25C pathway is upregulated. In addition, CDC25C activity is negatively regulated by ARID1A–ATR–CHK (checkpoint kinase) pathway under DNA damage conditions, thereby strictly controlling the CDC25 activity in ARID1A WT cells. In ARID1A MT cells, ARID1A–ATR–CHK pathways is impaired and thus CDC25C activity is de-repressed, causing it in hyper-active state. In this condition, cells can be addicted to AURKA–CDC25C pathway for cell survival and proliferation. Therefore, AURKA–CDC25C axis becomes a target for synthetic lethality in ARID1A- deficient cells

Article Snippet: Each aliquot of protein sample was run on a SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Santa Cruz, sc-32761, 1:1000 dilution), CDC25C (Santa Cruz, sc-327, 1:2000 dilution), CDC2 (Santa Cruz, sc-54, 1:2000 dilution), GAPDH (Santa Cruz, sc-365062, 1:5000 dilution), HSP90 (Santa Cruz, sc-69703, 1:5000 dilution), αtubulin (Santa Cruz, sc-5286, 1:5000 dilution), p-CDC25C (Cell Signaling, #9529s, 1:1000 dilution), p-CDC2 (Cell Signaling, #9111s, 1:1000 dilution), AURKA (Cell Signaling, #14475s, 1:2000 dilution), and cleaved caspase 3 (Cell Signaling, #9661s, 1:2000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).

Techniques: Western Blot, Inhibition, Phospho-proteomics, Expressing, Activity Assay, Mutagenesis

Journal: Cell Reports Methods

Article Title: Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

doi: 10.1016/j.crmeth.2021.100138

Figure Lengend Snippet:

Article Snippet: p38α(MAPK14) , Carna Biosciences , Catalog:04-152.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Sequencing, Modification, Software

Journal: Journal of Translational Medicine

Article Title: Vascular restenosis following paclitaxel-coated balloon therapy is attributable to NLRP3 activation and LIN9 upregulation

doi: 10.1186/s12967-024-05657-y

Figure Lengend Snippet: LIN9 binds to the promoter region of AURKA to increase its expression. A , B Western blotting and qRT-PCR experiments were conducted to analyze the effects of IL-1β and PTX on the protein and mRNA expression levels of AURKA and FOXM1 ( n = 3). C After knocking down LIN9, western blot experiments to observe whether the promoting effect of IL-1β on downstream molecules was blocked ( n = 3). D , E The effects of different drug treatments on the protein expressions of AURKA and FOXM1 were evaluated using western blot assay ( n = 3). F Primer design for AURKA was conducted based on the predicted binding sites, and ChIP followed by qRT-PCR experiments were performed to validate the enrichment levels of each binding site ( n = 3). G The relative luciferase activity of plasmids containing the wild-type AURKA promoter (WT) or its mutants was measured in 293T cells transfected with plasmids overexpressing LIN9 or control plasmids. The upper sequences show the LIN9 binding motif (blue) and its mutant (red; n = 3). H The differences in the enrichment levels of LIN9 at the AURKA promoter region were observed under different drug treatments ( n = 3). Data are presented as mean ± SEM (ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001). I The EMSA was performed to confirm the binding of LIN9 to the A means p (+ 34/+45) and B means p (+ 66/+77) binding sites of AURKA

Article Snippet: F Primer design for AURKA was conducted based on the predicted binding sites, and ChIP followed by qRT-PCR experiments were performed to validate the enrichment levels of each binding site ( n = 3).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay, Transfection, Control, Mutagenesis

Journal: Cancer Cell International

Article Title: Silencing of AURKA augments the antitumor efficacy of the AURKA inhibitor MLN8237 on neuroblastoma cells

doi: 10.1186/s12935-019-1072-y

Figure Lengend Snippet: AURKA knockdown induced cell apoptosis and cell growth inhibition by repressing the activity of Akt/Stat3 pathway ( a ) 2.0 × 10 5 of IMR32 cells were seed into 6-well plate. The next day, cells were transfected with AURKA siRNAs, siAURKA-1, si-AURKA-2, or siRNA CTRL. At 48 h after transfection, cells were harvested and total protein were extracted. Western blotting was performed for AURKA expression. b 2.0 × 10 5 of IMR32 cells were seed into 6-well plate. The next day, cells were transfected with AURKA siRNA-1. At 48 h after transfection, cells were harvested and cell apoptosis and cell cycle analysis ( c ) were was performed by flow cytometry. d 5 × 10 3 of IMR32 cells were seed into 96-well plate. The next day, cells were transfected with AURKA siRNA-1. At d0, d1,d2,d3,d4,d5,d6 time points, MTT assay was adopted for cell vialility test. Each sample was analyzed by triplicates. Error bars correspond to the averages ± S.D. e 2.0 × 10 5 of IMR32 cells were seed into 6-well plate. The next day, cells were transfected with AURKA siRNA-1. At 48 h after transfection, cells were harvested and western blot was performed for expression of AURKA, pAURKA, Akt, p-Akt, STAT3, p-STAT3, JAK2, and p-JAK2

Article Snippet: AURKA siRNAs were synthesized by RIBOBIO, and their sequences were as follows: AURKA siRNA-1: ATGCCCTGTCTTACTGTCA; AURKA siRNA-2: ATTCTTCCCAGCGCGTTCC. siN05815122147 NControl_05815 (standard) from RIBOBIO served as the siRNA control.

Techniques: Inhibition, Activity Assay, Transfection, Western Blot, Expressing, Cell Cycle Assay, Flow Cytometry, MTT Assay

Journal: eLife

Article Title: Aurora kinase A localises to mitochondria to control organelle dynamics and energy production

doi: 10.7554/eLife.38111

Figure Lengend Snippet:

Article Snippet: Antibody , AURKA , Merck Millipore , PC742 , 1:1000.

Techniques: Sequencing, Control, Recombinant, shRNA