Journal: Frontiers in oncology

Article Title: PDCD10-Deficiency Promotes Malignant Behaviors and Tumor Growth via Triggering EphB4 Kinase Activity in Glioblastoma.

doi: 10.3389/fonc.2020.01377

Figure Lengend Snippet: FIGURE 1 | Knockdown of PDCD10 increases the expression and the kinase activity of EphB4 in GBM cells and promotes cell growth, which is reversed by the treatment with the specific inhibitor of EphB4 kinase NVP. PDCD10 was knocked down in U87 or T98g cells by lentiviral shRNA transduction. (A) RT2-PCR demonstrated a significant downregulation of PDCD10 mRNA and an upregulation of EphB4 mRNA in PDCD10 knockdown (shPDCD10) U87 and T98g cells in comparison to EV-transduced cells. (B) ELISA demonstrated that knockdown of PDCD10 elevated the level of p-EphB4, reflecting the activity of EphB4, which was reversed upon the treatment with NVP (10 nM), a specific EphB4 kinase inhibitor. (C) Western blot revealed that knockdown of PDCD10 increased the EphB4 protein expression and activated Erk1/2 but not Akt in GBM cells (left). The activation of Erk1/2 mediated by PDCD10 knockdown was partially reversed by the treatment of NVP. Semiquantification of the blots was performed measuring the integrated optical density (IOD) of the blots normalized to the IOD of housekeeping protein GAPDH (right). (D) Knockdown of PDCD10 accelerated GBM cell growth. This effect was reversed by the treatment with NVP for 24 or 48 h as shown by fluorescence measurement of U87-(RFP) and T98g-(GFP) cells. For transduced U87 cells expressing RFP, the excitation wavelength was 553 nm and the emission wavelength was 574 nm. For transduced T98g cells expressing GFP, the excitation wavelength was 485 nm and the emission wavelength was 535 nm. All data presented in (A–D) were representative of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with EV; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with shPDCD10.

Article Snippet: The following antibodies were used: PDCD10 (1:400; Atlas Antibodies); EphB4 (1:400, Santa Cruz), p-Erk1/2, p-Akt, and GAPDH (each 1:1000 dilution, Cell Signaling Technology).

Techniques: Knockdown, Expressing, Activity Assay, shRNA, Transduction, Comparison, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay

Journal: Frontiers in oncology

Article Title: PDCD10-Deficiency Promotes Malignant Behaviors and Tumor Growth via Triggering EphB4 Kinase Activity in Glioblastoma.

doi: 10.3389/fonc.2020.01377

Figure Lengend Snippet: FIGURE 4 | Inhibition of EphB4 suppresses PDCD10-knockdown mediated EphB4 activation and the tumor growth in a human GBM xenograft model in mice. (A) Confirmation of the knockdown of PDCD10 mRNA (a) and protein (b) in U87 cells before the implantation. (B) Expression of PDCD10 in resected tumors at mRNA (a) and protein levels (b) determined by RT2-PCR and Western blot, respectively. (C) ELISA for p-EphB4 demonstrated the activation of EphB4 in PDCD10-knowckdown tumors, which was completely reversed after treatment with NVP. (D) The tumor growth curve revealed a significantly faster tumor progression in PDCD10-knockdown tumors and a marked tumor growth inhibition by the treatment with NVP. (E) NVP treatment reduced significantly the tumor mass compared to that from PDCD10-knowckdonw mice. Upper: the representative images show the tumors from control, shPDCD10, and NVP-treated shPDCD10-mice, respectively. Lower: statistical analysis, *P <0.05, **P < 0.01, ***P < 0.001, compare to control; #P < 0.05, ##P < 0.01, compare to shPDCD10 (n = 7, each group).

Article Snippet: The following antibodies were used: PDCD10 (1:400; Atlas Antibodies); EphB4 (1:400, Santa Cruz), p-Erk1/2, p-Akt, and GAPDH (each 1:1000 dilution, Cell Signaling Technology).

Techniques: Inhibition, Knockdown, Activation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Genetics in Medicine

Article Title: Janus-faced EPHB4 -associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes

doi: 10.1038/s41436-021-01136-7

Figure Lengend Snippet: Functional annotation of the thirteen EPHB4 variants investigated in this study.

Article Snippet: Fifty nanograms of the EPHB4 mammalian expression vector Myc-DDK-tagged pCMV6-Entry-EPHB4 (RC208559, OriGene) was used as template with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).

Techniques: Functional Assay

Journal: Genetics in Medicine

Article Title: Janus-faced EPHB4 -associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes

doi: 10.1038/s41436-021-01136-7

Figure Lengend Snippet: ( a ) Western blot analysis of EPHB4 expression detected with anti-DDK antibody and GAPDH used as a loading control. The position of molecular mass markers (in kDa) is indicated to the right of the gel. ( b ) Effect of EPHB4 variants on EPHB4 tyrosine phosphorylation in LECs after EphrinB2 stimulation with 1 μg/ml clustered Ephrin B2/Fc (EB2/Fc) or Fc alone. Receptor phosphorylation was analyzed by immunoprecipitation with anti-DDK antibody and western blotting using anti-p-tyrosine (upper panel) and EPHB4 (lower panel) antibodies. ( c ) Subcellular localization of EPHB4 variants. EPHB4 receptor was visualized with anti-DDK antibody (green), cell–cell contact with anti-VE-cadherin antibody (red) and nuclei with DAPI (blue). Membrane localization of the receptor is marked with arrowheads, cytoplasmic reticular localization with an asterisk, and intracellular aggregates with arrows. Images taken at 20× magnification with an EVOS™ M5000 imaging system. Scale bar 125 µm. For ( a – c ), one representative of ≥3 experiments is shown. ( d ) Model for potential EPHB4 molecular disease mechanisms. Left: Some mutant receptors were present in the membrane, lacking tyrosine activity. Whether this leads to EPHB4 haploinsufficiency or a dominant negative effect of the mutant EPHB4 was not tested in this study. Most variants fitting with this model were cases with a lymphatic-related fetal hydrops (LRFH) phenotype. Right: Other mutant receptors were sequestered into intracellular aggregates, leading to a loss-of-function disease mechanism. Most variants fitting with this model were cases with a CM-AVM2 phenotype. Bidirectional Eph-Ephrin signaling is complex with the existence of receptor–ligand oligomerization and bidirectional endocytosis of receptor–ligand complexes and we can only speculate on how forward or reverse signaling would be affected. MUT mutant EPHB4 receptor, WT wild-type EPHB4 receptor.

Article Snippet: Fifty nanograms of the EPHB4 mammalian expression vector Myc-DDK-tagged pCMV6-Entry-EPHB4 (RC208559, OriGene) was used as template with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).

Techniques: Western Blot, Expressing, Immunoprecipitation, Imaging, Mutagenesis, Activity Assay, Dominant Negative Mutation