Fig 2: Cinobufagin induces cell cycle arrest at G2/M phase. (A) Cell cycle distribution of Huh-7 cells following treatment with 5 µmol/l cinobufagin for 24 h, as determined using flow cytometry. (B) Cell cycle analysis of Huh-7 cells. (C) Protein expression levels of CDK1, cyclin B1 and PCNA following treatment with 5 µmol/l cinobufagin for 24 h. Densitometric analysis of (D) CDK1, (E) cyclin B1 and (F) PCNA. ß-actin was used as a control. Data are presented as the means ± standard error of the mean of three independent experiments. *P<0.05 vs. control, #P<0.05 vs. cinobufagin. AURKA, aurora kinase A; CDK1, cyclin-dependent kinase 1; PCNA, proliferating cell nuclear antigen.

Fig 3: In vitro and in vivo synthetic lethality in ARID1A-/- RKO cells by AURKA inhibition. a Stable expression of ARID1A in ARID1A-/- RKO cells using a lentiviral transduction. Three selected ARID1A stable clones are shown. b Dose–response curves of ARID1A-/- parental RKO and ARID1A overexpressing (ARID1AOE) RKO clones treated with AURKAi. Error bars represent s.d. (n = 6) from three independent experiments. Survival curve of ARID1A-/- versus ARID1AOE cell lines, P value < 0.0001, ANOVA. c Synthetic lethality effect of AURKA siRNA (siAURKA#2) on RKO ARID1A isogenic pair. Representative cell images were taken with IncuCyte ZOOM. Scale bars, 300 µm. d Integrated cell density was measured with the IncuCyte ZOOM software as a surrogate for cell viability (right panels). Error bars represent s.d. **P < 0.01, Student’s t test. e Schematic illustration of mouse tumor xenograft experiments with RKO ARID1A isogenic cell pair. f, g Tumor growth curve in nude mice bearing ARID1A-/- RKO (f) or ARID1AOE RKO clone #2 (g) xenografts after injection of vehicle or 60 mg kg-1 (mpk) AURKAi. Error bars represent s.d. *P < 0.05; **P < 0.01 between vehicle and AURKAi treatment groups (n = 6), Student’s t test. h, i Wet weight measurement of the tumors isolated from mice bearing ARID1A-/- RKO (h) or ARID1AOE RKO clone #2 (i) xenografts at 20 days after injection of vehicle, 30 or 60 mpk AURKAi. Error bars represent s.d. **P < 0.01 between vehicle and 60 mpk AURKAi treatment groups (n = 6), Student’s t test

Fig 4: Primaquine reduced the interaction of nEGFR and Stat3 and reduced the transcript and protein levels of c-Myc. (A) nEGFR and Stat3 were immunoprecipitated from the nuclear protein extracts of breast cancer cells under primaquine treatment with anti-EGFR or anti-Stat3 antibodies. Western blotting was performed using the indicated antibodies. (B) The target genes of the Stat3/nEGFR complex were Stat1, Aurora-A, COX2, and c-Myc. (C) Effect of primaquine on transcript levels, total levels, and nuclear levels of the Stat1, Aurora-A, COX2, and c-Myc proteins. Total and nuclear c-Myc protein levels under primaquine (50 and 80 µM) or DMSO treatment conditions. The data are presented as the mean ± SD; n = 3; * indicates p < 0.05 vs. control. (D) siRNA-induced silencing of Stat3 reduced the transcript and protein levels of c-Myc. Representative images of Western blots are shown. Data from triplicate experiments are shown as the mean ± SD. Compared with control, * indicates p < 0.05. (E) Inhibition of nEGFR function by primaquine. EGF stimulation induces EGFR dimerization and internalization to endocytic vesicles. EGFR then undergoes nuclear translocation. EGFR is consistently detected in the nuclei of breast cancer cells. nEGFR binds to Stat3, and the EGFR/Stat3 complex regulates the transcription of Stat1, Aurora kinase A, COX2, and c-Myc. Primaquine reduces nEGFR expression by inhibiting EGFR trafficking.

Fig 5: WDR62 mediates the stability of the TPX2/AURKA protein complex in prostate cancer.A and B Scatter plots (n = 99) showing a correlation between TPX2 and WDR62 and AURKA and WDR62, respectively5. Spearman’s correlation with a two-tailed test for significance was calculated; C Co-immunoprecipitation of WDR62 with TPX2 and AURKA (* non-specific band). The co-immunoprecipitation experiment was performed twice to determine reproducibility; D Western blot showing loss of AURKA and TPX2 following knockdown of WDR62. Each western blot experiment was performed twice to determine reproducibility; E Western blot of AURKA following knockdown of WDR62 with and without MG132, a proteasome inhibitor. Each western blot experiment was performed twice to determine reproducibility; F and G Scatter plots showing phenotype (gene effect) correlation between TPX2 and AURKA and TPX2 and WDR62, respectively14. Spearman’s correlation was performed for statistical analysis.