Fig 2: MELK is crucial for regulating E2F8 expression in prostate cancer cells. (A) Pearson correlation analysis showed coexpression correlation between MELK and E2F8 in TCGA-PRAD and cBioPortal transcriptomic data. (B) MELK expression levels in patients with prostate cancer (GSE3325) were represented in the box plots. ***P<0.005, n.s., not significant. (C) Each cell was transfected with 50 nM siMELK-1 or siMELK-2 for 48 h prior to western blot analysis. (D) Each cell was transfected with 50 nM siMELK-1 for 9 days (once every 3 days), after which colony formation assessed. The data were expressed as the mean ± SEM (n=3). **P<0.01; ***P<0.005. TCGA-PRAD, The Cancer Genome Atlas-Prostate Adenocarcinoma; r, Pearson correlation coefficient; si, short interfering.

Fig 3: CsA downregulates E2F8 expression in prostate cancer cells. (A) PC-3 and LNCaP cells were treated with 10 µM CsA for the indicated times prior to RT-PCR and western blot analysis. (B) PC-3, LNCaP, DU145 and 22Rv1 cells were treated with the indicated concentrations of CsA for 48 h prior to RT-PCR and western blot analysis. (C) Each cell was transfected with 50 nM siE2F8-1 or siE2F8-2 for 48 h prior to MTT assay. Cell viability was expressed as a relative value compared with that of siControl which was set to 100%. The data were expressed as the mean ± SEM (n=6) ***P<0.005. (D) Each cell was transfected with 50 nM siE2F8-1 for 9 days (once every 3 days), after which colony formation assessed. The data were expressed as the mean ± SEM (n=3) **P<0.01, ***P<0.005. CsA, cyclosporin A; RT-PCR, reverse transcription PCR; si, short interfering.

Fig 4: Clinical significance of E2F8 in prostate cancer. (A and C) Coexpression correlation between E2F8 and AR or between MELK and AR in TCGA-PRAD and cBioPortal transcriptomic data. (B and D) Survival curve for patients with prostate cancer based on the expression levels of E2F8 and AR or those of MELK and AR from cBioPortal data. (E) LNCaP cells were treated with 50 nM siE2F8-1, 10 µM MDV300, or both for 72 h prior to MTT assay. The data were expressed as the mean ± SEM (n=4-6). ***P<0.005. (F) LNCaP cells were treated with 50 nM siMELK-1, 10 µM MDV300, or both for 72 h prior to MTT assay. The data were expressed as the mean ± SEM (n=4-6) ***P<0.005. (G) Inferred TF motif activity differences between patients with CR (highlighted in blue) and SD (highlighted in red). The x-axis denotes the mean TF activity differences and the y-axis indicates-log10 (P-values). The vertical dotted line represents an absolute mean TF activity difference of 0.008 and the horizontal dotted line means the P-value 0.05 for significant TFs. AR, androgen receptor; TCGA-PRAD, The Cancer Genome Atlas-Prostate Adenocarcinoma; r, Pearson correlation coefficient; TF, transcription factor; si, short interfering; TCGA-PRAD, The Cancer Genome Atlas-Prostate Adenocarcinoma.