Fig 2: Schematic diagram of the cMELK-mediated pathway in GBM cellscMELK upregulation increased the adsorption of miR-593, which increases the degradation of EphB2 and thus promotes GBM mesenchymal transition and GSC maintenance.

Fig 3: cMELK promotes mesenchymal transition and GSC maintenance of GBM cells through the miR-593/EphB2 pathway(A) qRT-PCR assays detected the expression levels of EphB2 in the U87 and pGBM-1 cells upon cMELK knockdown alone or silencing miR-593 alone or in combination. (B) Western blot analysis of EphB2 in the indicated cells, GAPDH served as the loading control. (C and D) Representative images and quantification of Matrigel invasion for transfected U87 and pGBM-1 cells. Scale bar, 100 μm. (E and F) Representative images and quantification of three-dimensional spheroid assays for transfected U87/GFP tumor-spheres and pGBM-1/GFP tumor-spheres. Scale bar, 50 μm. (G) Representative photomicrographs of new clonal sphere after transduced. (H) The statistical analysis of the percentage of positive wells of clonogenic assay were measured the stemness of GSCs after transduced. Scale bar, 50 μm. (I and J) The cellular effects of transduced U87-GSCs and pGBM-1-GSCs were assessed by CellTiter-Glo assay and direct cell count. Data are presented as the means of triplicate experiments. Student’s t test was used (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Fig 4: EPHB2 promotes DEPTOR tyrosine 289 phosphorylation and mTOR activation.A, 293T cells were transfected with FLAG-DEPTOR-D4 in combination with HA-GFP, EPHA1-GFP, EPHA2-GFP, EPHA3-GFP, EPHB1-GFP, EPHB2-GFP, EPHB3-GFP, or EPHB4-GFP. Cells were lysed for a denaturing FLAG-tag immunoprecipitation to reveal the levels of tyrosine phosphorylation of DEPTOR. B, 293T cells and EPHB2-depleted 293T cells were transfected with FLAG-DEPTOR-D4 and lysed to perform a FLAG-denaturing immunoprecipitation to reveal the levels of DEPTOR-D4 tyrosine phosphorylation. C, EPHB2-depleted 293T cells were transfected with FLAG-DEPTOR-D4 in combination with EPHB2WT or EPHB2KD (kinase dead). Cells were lysed to perform a FLAG-denaturing immunoprecipitation to reveal the levels of DEPTOR-D4 tyrosine phosphorylation. D, 293T cells transfected with FLAG-DEPTOR-D4 in combination with EPHB2WT were treated with PP242 (10 μM) or PP2 (10 μM) for 2 h before cells lysis. FLAG-denaturing immunoprecipitation to reveal the levels of DEPTOR-D4 tyrosine phosphorylation. E, lysate from EPHB2-depleted or EPHB2WT-overexpressing HeLa cells were used to assess mTOR activity. HA, hemagglutinin; mTOR, mechanistic target of rapamycin.

Fig 5: Model of mTOR activation by EPHB2-regulated DEPTOR tyrosine 289 phosphorylation. Binding of ephrins to EPHB2 receptors causes their clustering and activation. Active EPHB2 receptors phosphorylate the tyrosine kinase SYK, causing its activation and the phosphorylation of DEPTOR on its tyrosine 289. This latter phosphorylation event increases DEPTOR stability, whereas decreasing its affinity to mTOR, leading to an increased and sustained activation of mTOR. Conversely, inactivated EPHB2 receptors limit DEPTOR tyrosine 289 phosphorylation, which allows DEPTOR binding to mTOR, thus inhibiting its activity. mTOR, mechanistic target of rapamycin; SYK, spleen tyrosine kinase.