Fig 2: TOPK depletion causes multinucleation and apoptosis in HCT116 cells.Cells were irradiated 72 h post siRNA treatment. (A) Live-cell imaging following the fate of 50 HCT116 H2B-mCherry cells for 48 h after 4 Gy IR. Each horizontal bar represents one cell, with interphase shown in grey and mitosis shown in black. Following division, the first daughter cell to undergo division, apoptosis or multinucleation is shown. (B) The percentage of cells that apoptosed or became multinuclear during the first or second cell divisions after IR (n=3, average of 3 replicates). (C) Analysis of IR-induced multinucleation in fixed HCT116 treated cells 72 h after 4 Gy IR. At least 100 cells were counted per condition. Nuclei were stained with the DAPI DNA stain (blue) and FITC was used as a cytoplasmic counterstain (green). (D) Cell death in the presence (right panel) and absence (left panel) of 4 Gy irradiation was assessed with Hoechst staining to identify bright apoptotic nuclei in cells transfected with NT or TOPK siRNA and irradiated with 4 Gy at T=0. Data displayed are representative of three independent experiments and were analysed using unpaired two-sided t-tests; *P<0.05, **P<0.01 and ***P<0.001.

Fig 3: PBK is a target gene of miR-216b-3p. (A) TargetScan was applied to predict the interaction between miR-216b-3p and 3′UTR of PBK. (B) Luciferase activity was detected via a dual luciferase assay. (C) Relative miR-216b-3p expression levels in GLC-82 cells. (D) Effects of miR-216b-3p on PBK/TOPK protein expression in GLC-82 cells were determined by western blotting. (E) Effects of miR-216b-3p on PBK/TOPK mRNA expression in GLC-82 cells were determined by reverse transcription-quantitative polymerase chain reaction. (F) Relative PBK/TOPK mRNA expression levels in GLC-82 cells. Data are presented as the mean ± standard deviation. **P<0.01 vs. control. Control, control group, cells without any treatment; NC, negative control group, cells transfected with the negative control vector of miR-216b-3p mimics; mimic, cells transfected with miR-216b-3p mimics; plasmid, cells transfected with PBK/TOPK plasmids; LUC, luciferase; miR, microRNA; PBK/TOPK, PDZ-binding-kinase/T-LAK cell-originated protein kinase; MUT, mutated 3′-untranslated region; WT, wild type 3′-untranslated region.

Fig 4: Acetylshikonin attenuates the expression of proteins involved in TOPK signalling. (a) The effect of acetylshikonin on TOPK signalling in colon cancer cells was assessed by western blot analysis followed by densitometric quantification. The cells were treated with 0‐, 5‐, or 10‐μM acetylshikonin and harvested at 24 hr, and then cell lysates were subjected to western blotting. Data shown are individual values with means± SD; n=5 independent experiments. *P < .05, significantly different as indicated. (b) The effect of acetylshikonin was evaluated in colon cancer cells transfected with an NF‐κB luciferase reporter plasmid. Data shown are individual values with means± SD; n=5 independent experiments. Data shown are individual values with means± SD; n=5 independent experiments

Fig 5: High TOPK expression correlates with poor outcomes in prostate cancer patients.(A) HCT116 cells were transfected with siRNA and irradiated (4 Gy). Metaphases were collected at 24, 48 and 72 h following irradiation and scored for chromosomal aberrations, with 40 nuclei analysed in each group. Images of representative metaphases include examples of typical errors detected (insets). Arrows indicate chromosomal aberrations. (B) Average number of errors per metaphase from three independent experiments *P<0.05 and **P<0.01. (C) Representative photomicrographs taken following immunohistochemistry staining with an anti-TOPK antibody, showing non-staining in disease-free prostate epithelium and strong nuclear and cytoplasmic TOPK expression in prostatic carcinoma. (D) Kaplan–Meier analysis of TOPK expression and overall survival, and any recurrence in prostate cancer patients following radical radiotherapy (n=128). P-value from univariate analysis.