Journal: bioRxiv

Article Title: AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

doi: 10.1101/2025.07.29.665837

Figure Lengend Snippet: Shown is mRNA expression of AURKA (A), CDKN2A (B), PPP2R4 (C) and of an AURKA signature (D) consisting of expression of AURKA and 18 genes important for regulating AURKA during progression of NB in ganglia of TH-MYCN mice compared to ganglia from wild-type mice. The means and standard deviations of four samples depending on age are depicted. The difference in the regression slope between transgenic and wild-type samples was calculated. The multiple testing-corrected p -value for the linear regression analysis is shown. E) Enhanced expression of AURKA after transfection and stable selection of SH-EP-MYCN-ER cells. Representative Western blot of AURKA in transfected SH-EP-MYCN-ER cells. AURKA expression was quantified by densitometry relative to β-Actin. F) Enhanced anchorage-independent growth of SH-EP-MYCN-ER cells overexpressing AURKA upon induced nuclear translocation of MYCN-ER. Stably selected SH-EP-MYCN-ER cells were seeded into soft agar in 24-well plates and grown for 2 weeks. 4-hydroxytamoxifen (4-OHT) was added to culture media to translocate MYCN-ER into the nucleus. Colonies of more than 30 cells were counted. Means of more than 20 wells are shown. ***, p < 0.001 and **** p < 0.0001 by one-way ANOVA.

Article Snippet: Membranes were probed with the primary antibodies rabbit anti-human AURKA (#4718, 1:1000, Cell Signaling Danvers, MA), rabbit anti-human-PPP2R4 (PTPA) (#HPA005695, 1:5000, Sigma-Aldrich), mouse anti-mePPP2CA (sc-81603, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Actin (A5441, 1:5000, Sigma-Aldrich) and mouse anti-Tubulin (clone DM1A1, 1:5000, Sigma-Aldrich).

Techniques: Expressing, Transgenic Assay, Transfection, Selection, Western Blot, Translocation Assay, Stable Transfection

Journal: bioRxiv

Article Title: AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

doi: 10.1101/2025.07.29.665837

Figure Lengend Snippet: Kaplan-Meier analysis of overall survival of 498 clinically annotated NB patients (SECQ-GSE62564) depending on transcript levels of AURKA , PPP2R4 and PPP2CA (A) or of the combination of AURKA and PPP2R4 or AURKA and PPP2CA (B) are shown. Statistical analysis of Kaplan-Meier curves was performed using the log-rank test with Bonferroni correction. The cut-off was determined by the scanning method.

Article Snippet: Membranes were probed with the primary antibodies rabbit anti-human AURKA (#4718, 1:1000, Cell Signaling Danvers, MA), rabbit anti-human-PPP2R4 (PTPA) (#HPA005695, 1:5000, Sigma-Aldrich), mouse anti-mePPP2CA (sc-81603, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Actin (A5441, 1:5000, Sigma-Aldrich) and mouse anti-Tubulin (clone DM1A1, 1:5000, Sigma-Aldrich).

Techniques:

Journal: bioRxiv

Article Title: AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

doi: 10.1101/2025.07.29.665837

Figure Lengend Snippet: A) Bayesian analysis of mRNA expression in ganglia and NB of TH-MYCN mice suggests a role of PPP2R4 in dysregulation of AURKA in neuroblastomagenesis. Microarray mRNA data previously described in were used. 85 genes implicated in the AURKA network were subjected to Bayesian analysis. The direction (arrows) and probability (edge thickness) of influence of the top 20 genes according to the differences of mean expression in NB vs. ganglia are shown. Genes are color-coded according to function in relation to AURKA. B) Loss of PPP2R4 depletes mePPP2CA protein and increases AURKA protein in KELLY cells. KELLY cells were transiently transfected with a CRISPR/Cas9 construct targeting PPP2R4 , sorted and seeded as single cells. Single-cell clones were expanded and classified as PPP2R4 wild-type or homozygous knockout. Shown are Western blots of PPP2R4, methylated PPP2CA (mePPP2CA), AURKA and Tubulin as loading control. C) Schematic cartoon depicting the relationship between AURKA, PPP2R4, MYCN and INK4A/ARF in promoting malignant properties of NB.

Article Snippet: Membranes were probed with the primary antibodies rabbit anti-human AURKA (#4718, 1:1000, Cell Signaling Danvers, MA), rabbit anti-human-PPP2R4 (PTPA) (#HPA005695, 1:5000, Sigma-Aldrich), mouse anti-mePPP2CA (sc-81603, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Actin (A5441, 1:5000, Sigma-Aldrich) and mouse anti-Tubulin (clone DM1A1, 1:5000, Sigma-Aldrich).

Techniques: Expressing, Microarray, Transfection, CRISPR, Construct, Clone Assay, Knock-Out, Western Blot, Methylation, Control

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, Control, Knockdown, 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, Control, Knockdown, shRNA, Mutagenesis, Activity Assay