Fig 2: NUAK1 regulation of PLK4 is mediated by GSK3β. (A) Quantification of centrosome number by γ‐tubulin IF staining in synchronised Mia PaCa‐2 cells transfected with MYPT1, or non‐targeting (siC), siRNAs. Fifty cells scored per condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc t‐test. (B) Immunoblot of total PLK4 in Mia PaCa‐2 cells transfected with MYPT1, versus non‐targeting (siCtrl), siRNA. Representative of three independent experiments. (C) Immunoblot of Ser9 phospho‐GSK3β in Mia PaCa‐2 cells transfected with NUAK1, versus non‐targeting, siRNA. Representative of three independent experiments. (D) Immunoblot of Ser9 phospho‐GSK3β in Mia PaCa‐2 cells treated with 10 μm HTH‐01‐015 or DMSO vehicle for 4 h. Representative of three independent experiments. (E) Immunoblot of total PLK4 in Mia PaCa‐2 cells transiently transfected with constitutively active (S9A) or kinase‐dead (K85R) mutant GSK3β expression vector, compared with empty vector (EV). Representative of three independent experiments. (F) Immunoblots for endogenously expressed PLK4, NUAK1, and GSK3β in lysates and anti‐PLK4 immunoprecipitates, or IgG control IPs, of untreated asynchronous Mia PaCa‐2 cells. Representative of two independent experiments. (G) Immunoblot of total PLK4 in Mia PaCA‐2 cells pre‐treated for 1 h with 3 μm CHIR‐99021 (GSK3i) or DMSO vehicle, followed by 10 μm HTH‐01‐015 ± 3 μm CHIR‐99021 for 1 h immediately prior to harvest. Representative of three independent experiments. (H) Quantification of centrosome number by γ‐tubulin IF in mitotic Mia PaCa‐2 cells treated with CHIR‐99021 (GSK3i), HTH‐01‐015 (HTH), both, or vehicle ctrl, from time of release from thymidine block. Cells were fixed for analysis at 10.5 h post‐release. Fifty cells were scored per treatment condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. (I) Quantification of incidence of nuclear aberrations in Mia PaCa‐2 cells treated with CHIR‐99021 (GSK3i), HTH‐01‐015 (HTH), both, or vehicle ctrl, from time of release from thymidine block. HTH‐01‐015 and ctrl values are the same as Fig. 4F as experiment was performed simultaneously. Cells were fixed for analysis at 13 h post‐release. One hundred cells were scored per treatment group per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. For all panels, P value; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: PLK4 expression in HCoEpic and CRC cells. (A) Representative images of PLK4 detection by western blot. (B) Comparison of PLK4 expression between HCoEpic and CRC cells. One-way ANOVA followed by Dunnett's multiple comparisons test was applied among HCoEpic, HT-29, HCT-116, LoVo and SW480 cells. PLK4, polo-like kinase 4; CRC, colorectal cancer.

Fig 4: Effect of PLK4-siRNA on 5-FU sensitivity in CRC cell lines. (A) Comparison of the mRNA expression of PLK4 among blank control, NC-siRNA and PLK4-siRNA-treated HCT-116 cells. One-way ANOVA followed by Tukey's multiple comparisons test was applied. (B) Comparison of cell viability among blank control, NC-siRNA and PLK4-siRNA-treated HCT-116 cells groups. One-way ANOVA followed by Dunnett's multiple comparisons test was applied. (C) Changes in the IC50 value of 5-FU between NC-siRNA and PLK4-siRNA-treated HCT-116 cells. Student's t-test was applied. (D) Comparison of the mRNA expression of PLK4 among blank control, NC-siRNA and PLK4-siRNA-treated LoVo cells. One-way ANOVA followed by Tukey's multiple comparisons test was applied. (E) Comparison of cell viability among blank control, NC-siRNA and PLK4-siRNA-treated LoVo cell groups. One-way ANOVA followed by Dunnett's multiple comparisons test was applied. (F) Changes in the IC50 value of 5-FU between NC-siRNA and PLK4-siRNA-treated LoVo cells. Student's t-test was applied. PLK4, polo-like kinase 4; 5-FU, 5-fluorouracil; CRC, colorectal cancer; IC50, 50% inhibitory concentration; siRNA, short interfering RNA; NC, negative control.

Fig 5: Effect of PLK4-siRNA on β-catenin nuclear translocation in CRC cell lines. (A) Detection of β-catenin in the nuclei of CRC cells. (B) Comparison of β-catenin in the nuclei of CRC cells. One-way ANOVA followed by Tukey's multiple comparisons test was applied. PLK4, polo-like kinase 4; CRC, colorectal cancer; siRNA, short interfering RNA; NC, negative control.