Fig 2: MLK3 deficiency attenuates glioblastoma multiforme (GBM) cell migration and invasion and disrupts actin cytoskeleton rearrangement. (A) MLK3 proteins are overexpressed in highly migrating GBM cells. Cells were treated with serum-free DMEM for 48 h. Immunoblot analysis of MLK3 levels in several GBM cell lines. GAPDH was used as a loading control. (B, C) Immunoblot assay of the MAP3K11 gene knockout in U251 (B) and U118 (C) cells. GAPDH was used as a loading control. (D–G) Transwell assay of GBM cell migration and invasion in MAP3K11 gene knockout U251 (D, F) and U118 (E, G) cells. Two-tailed Student’s t-test. Scale bars, 100 μm; n = 3; **P < 0.01. (H, I) The loss of MLK3 disrupts actin cytoskeleton rearrangement. The results shown are representative of at least three independent cultures. Phalloidin was used to label F-actin (red), anti-vinculin antibody was used to stain focal adhesion structure (green), and DAPI was used to stain nuclei (blue). White arrows indicate the actin based “mass-like structures.” Scale bars, 20 μm.

Fig 3: MLK3 directly binds with EPS8. The results shown are representative of at least three independent experiments. (A, B) Overexpressed and endogenous MLK3 binds to EPS8 in U251 cells. U251 cells were cotransfected with pcDNA3.1-Myc-MLK3 and pcDNA3.1-His-EPS8 plasmids for 24 h, with the pcDNA3.1 plasmid as a control. The lysates were immunoprecipitated using anti-Myc (A), anti-EPS8 (B) or control IgG, EPS8, or MLK3 was detected with an immunoblot assay. (C) The molecular structure of MLK3 and EPS8. (D) AAs 1–104 and 632–847 of MLK3 directly bound to EPS8 in vitro. GST-tagged MLK3 segments were expressed in BL21 cells. The purified proteins were incubated with the protein lysates from pcDNA3.1-EPS8-transfected HEK293 cells. The proteins were immunoblotted with indicated antibodies. * the target protein. (E) The expression of MLK3 and EPS8 predicts the overall survival of patients. The data were derived from the CGGA dataset (n = 286). Kaplan-Meier survival analyses for MLK3 and EPS8 mRNA levels. Log-rank test. *P < 0.05, **P < 0.01.

Fig 4: MLK3 regulates the localization of EPS8 in U251 and U118 cells. (A, B) The MAP3K11 gene knockout increases the expression of EPS8 in U251 (A) and U118 (B) cells. Relative levels of MLK3 were normalized to U251 wt or U118 wt groups. GAPDH was used as a loading control. Two-tailed Student’s t-test. n = 3, *P < 0.05, **P < 0.01. AU, arbitrary unit. (C, D) The MAP3K11 gene knockout alters the location of EPS8 in U251 (C) and U118 (D) cells. The results shown are representative of at least three independent cultures. EPS8 (green); DAPI-stained nuclei (blue). Scale bars, 20 μm.

Fig 5: High MLK3 levels are correlated with poor prognosis in patients with IDH-wt GBM. (A, B) High levels of MLK3 mRNA are prevalent in IDH-wt GBM. The frequencies of high MLK3 mRNA levels in IDH-wt gliomas (n = 136), primary GBM (n = 71), recurrent GBM (n = 15), and secondary GBM (n = 9) were analyzed. The data were derived from the CGGA dataset. The median level of MLK3 mRNA in glioma samples was chosen as the cut-off point. pGBM, primary GBM; rGBM, recurrent GBM; sGBM, secondary GBM. (C) MLK3 abundantly expressed in IDH-wt GBM. The percent of high MLK3 protein levels in IDH-wt gliomas (n = 57) was investigated. The data were obtained from IHC analysis. (D, E) High levels of MLK3 mRNA are associated with the poor prognosis of patients. Kaplan-Meier survival analyses for MLK3 mRNA expression (n = 286) (D) and MLK3 mRNA expression and IDH gene status (n = 286) (E). The data were derived from the CGGA dataset. Log-rank test. NS, not significant. *P < 0.05, **P < 0.01.