Fig 2: PLK4 is transcriptionally regulated by ATAD2 in GBM. (A) Top 20 most correlated genes to PLK4 expression in the TCGA database. Pearson's correlation analysis was performed. (B) RT-qPCR indicated that PLK4 expression was markedly increased following exogenous ATAD2 overexpression in U87 cells. *P<0.05, **P<0.01; t-test. (C) Luciferase assays showed that PLK4 promoter activity was increased following lentivirus-mediated overexpression of ATAD2 in U87 cells. ***P<0.001; one-way analysis of variance followed by Dunnett's post hoc test. ATAD2, ATPase family AAA domain-containing protein 2; GBM, glioblastoma; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; ns, not significant; PLK4, polo-like kinase 4; UTR, untranslated region; EV, empty vector.

Fig 3: PLK4 induces radioresistance in GBM. (A) RT-qPCR and (B) western blot analysis for PLK4 expression in U87 cells treated with or without radiation (12 Gy; *P<0.05, **P<0.01 vs. 0 h; one-way ANOVA followed by Dunnett's post hoc test. β-actin served as the control. (C) RT-qPCR results showed that PLK4 mRNA expression was markedly increased following radiation (12 Gy), and could be partially eliminated by shPLK4. **P<0.01, ***P<0.001; one-way ANOVA followed by Dunnett's post hoc test. (D) In vitro cell proliferation assay for U87 cells transduced with either shNT or shPLK4 lentiviruses, followed (or not) by radiotherapy (12 Gy). **P<0.01 vs. shNT; one-way ANOVA followed by Dunnett's post hoc test. (E) Flow cytometry (Annexin V and propidium iodide) determined the apoptosis of U87 cells transduced with shNT or shPLK4, followed (or not) by radiotherapy (12 Gy). (F) RT-qPCR analysis of U87 cells transduced with PLK4 overexpression or control lentivirus. **P<0.01; t-test. (G) Flow cytometry determined the apoptosis of U87 cells transduced with PLK4 overexpression or control lentivirus, followed (or not) by radiotherapy (12 Gy). GBM, glioblastoma; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; ns, not significant; PLK4, polo-like kinase 4; sh, small hairpin RNA; IR, radiation.

Fig 4: PLK4 promotes GBM proliferation and tumorigenesis. (A) Phase-contrast and fluorescence images showing that pGFP-shPLK4 lentivirus was successfully transduced into U87 cells. (B) RT-qPCR and (C) western blot analysis of U87 cells transduced with shPLK4 or shNT (***P<0.001 vs. shNT; t-test). β-actin served as the control. (D) In vitro cell proliferation assays showed that shPLK4 reduced U87 cell proliferation in cells (**P<0.01 vs. shNT; one-way ANOVA). (E) Representative images of hematoxylin and eosin-stained mouse brain sections implanted with U87 cells transduced with shPLK4 or shNT. (F) Kaplan-Meier analysis of nude mice intracranially implanted with transduced U87 cells (n=5; P=0.0244; log-rank test). GBM, glioblastoma; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; PLK4, polo-like kinase 4; sh, small hairpin RNA; NT, non-targeting control.

Fig 5: PLK4 is overexpressed in GBM. (A) Genome-wide transcriptome microarray analysis indicated that PLK4 was one of the seven overlapping gene candidates upregulated in GBM, as compared with normal astrocytes and in GBMs treated with or without radiotherapy. (B) Analysis of the TCGA database showed that PLK4 was highly expressed in all four subtypes of GBM. *P<0.05 and **P<0.01 vs. normal; one-way ANOVA followed by Dunnett's post hoc test. (C) RT-qPCR and (D) western blotting showed that PLK4 was overexpressed in the three GBM cell lines (U87, U138 and U251), compared with normal astrocytes (***P<0.001, one-way ANOVA followed by Dunnett's post hoc test). β-actin served as the control. GBM, glioblastoma; ANOVA, analysis of variance; RT-qPCR, reverse transcription quantitative polymerase chain reaction; PLK4, polo-like kinase 4; IR, radiation.