Fig 2: MELK plays an oncogenic role in NB(A) MELK protein levels were examined by immunoblotting assay in NB cell lines listed. (B) Effects of MELK knockdown on p-Rb levels in NB cell lines were shown by immunoblotting assay. (C) The effect of MELK knockdown on NB colony formation potential were shown.

Fig 3: MELK inhibitor OTSSP167 inhibits cell proliferation and induces apoptosis in NB(A) The effects of OTSSP167 on the proliferation of a panel of six NB cell lines using cck-8 assay were shown. (B) IC50 calculation of cell lines showen in (A) by using PRISM 5.0. (C) Effects of OTSSP167 on NB cell morphology were shown. (D) Effects of OTSSP167 on PARP and Caspase-3 cleavages in NB cells were shown by immunoblotting assay.

Fig 4: MELK is a prognostic indicator in NB(A and B) Kaplan-Meier curves show the probability of overall survival based on MELK expression level of 498 patients in the SEGC dataset (A) and 649 patients in the Kocak dataset (B). (C and D) R2-tumor neuroblastoma SEGC dataset (C) and Kocak dataset (D) show higher MELK expression level in MYCN-amplified cases in comparison to MYCN-non-amplified cases. (E) The Kocak dataset shows significant correlations of MELK with MYCN expression levels. (F and G) R2-tumor neuroblastoma SEGC dataset (F) and Kocak dataset (G) show correlation of MELK expression level with NB disease stages.

Fig 5: MELK is a transcription target of MYCN/MYC in NB(A) Stable sh-RNA-mediated knockdown of MYCN/MYC. The qPCR analyses of MYCN/MYC and MELK expression levels were shown. (B) Total proteins of MYCN/MYC and MELK were measured in the indicated cells. (C and D) MYCN ChIP-seq analysis showed binding peaks of MYCN on MELK 5’UTR. TSS- Translation Start Site. Designed primers for ChIP-seq analysis is shown with arrows under the peaks. These primers encompass the E-Box motif. (E) ChIP-qPCR analysis for MYCN binds on MELK 5’UTR site in MYCN3 cell line. Fold change with or without doxycycline treatment on MYCN3 cell line was shown. (F) ChIP-qPCR analysis for MYCN binds on MELK 5’UTR site in IMR-32 NB cell line. IP-IgG and IP-MYCN of MELK-ChIP assay on IMR-32 cells were shown. (G) qPCR analysis of MYCN/MYC and MELK mRNA levels in NB cell lines with stable MYCN/MYC-overexpression were shown. (H) MYCN/MYC and MELK protein levels in NB cell lines with stable MYCN/MYC-overexpression were shown.