PMC

Immunofluorescence of endogeneous expression of Evi-YFP in ESC colonies.Relative mRNA expression levels of Evi were analysed by quantitative RT-PCR. Evi-ESCs expressed increased Evi levels.Western blot of Evi overexpressing ESCs. Evi-ESCs expressed Evi-YFP fusion protein.Evi-ESCs stably transfected with 7TCF Firefly luciferase reporter showed increased Wnt reporter activity (*p < 0.05).Transfection of 7TCF Firefly luciferase reporter containing HEK293 cells with Evi-GFP plasmid led to increased Wnt reporter activity compared to transfection with GFP control vector (**p < 0.01).

Viability of RNAi transduced U87MG cells, A172, U251MG cells and T98G was determined by CellTiter-Glow assay and revealed reduced viability of Evi-RNAi transduced cells compared to control cells. β-catenin silencing had significant effect on viability of U787MG and U251MG cells. Evi and β-catenin silencing had no significant effect on proliferation of T98G cells (*p < 0.05).Evi and β-catenin silencing caused reduced colony formation in U251MG cells. Representative example of three-independent experiments is shown (right). Data are expressed as mean ± SD of three-independent experiments (*p < 0.05).

Neurosphere shape and size was disturbed after Evi silencing. Scale bar: 100 µm.Reduction in cell number compared to control transfected spheres 7 days after infection (**p < 0.01; ***p < 0.001).Lentiviral shRNA silencing of Evi expression in NCH421k and NCH644 cells led to an increase in the sub-G1 fraction (*p < 0.05; **p < 0.01).Representative graphs of cell cycle distribution. Data are expressed as mean ± SD of three-independent experiments.

Evi shRNA U87MG cells showed less transwell migration compared to control. Similar effect was achieved by siRNA transfection.Migration experiments were done as short-term assays to exclude anti-proliferative effects. Values represent mean ± SD from three-independent experiments (*p < 0.05; **p < 0.01).Reduced in vivo growth of glioma cells transfected with shRNA targeting Evi. The appearance of U87MG glioma xenografts formed by Evi shRNA or control transfected cells was reduced in the Evi downregulated glioma cells.

Western blot of U87MG cell lysates after RNAi transfection against Evi (Evi #1 and Evi #3) showed increased levels of phosphorylated STAT3 compared to control transfection. β-Actin was detected as loading control. Representative example of three-independent experiments is shown.Model of the Evi-Wnt-STAT3-IL8 signalling link in human glioblastoma cells. Evi mediated Wnt secretion controls phosphorylation of STAT3. Downregulation of Evi leads to activation of STAT3 by phosphorylation. Phosphorylated STAT3 binds to IL8 promotor and represses IL8 transcription. Downregulation of IL8 reduces glioblastoma cell proliferation and invasiveness.

Heatmap based on normalized U87MG gene expression values of RNAi Evi#1, Evi#3, β-catenin and control samples. Rows represent genes, which are differentially expressed (p < 0.01) and have an absolute log₂-fold change >1.5 compared to control, in Evi#1, Evi#3 or β-catenin silencing experiments.Over-represented KEGG categories in set of differentially expressed genes (Fisher's exact test, p < 0.01). The length of the bars represents the number of genes within the set of differentially expressed genes that are annotated as part of the corresponding KEGG category. The numbers is red indicate the significance of the over-representation. The KEGG categories are not mutually exclusive. ‘Cell cycle’ is the most significant over-represented category.Cells were transduced with indicated siRNAs. The relative mRNA expression levels of Evi, β-catenin, cyclin D1, c-Myc, PTMA, tenascin-C, and IL8 after Evi silencing analysed by quantified RT-PCR. Data are expressed as mean ± SD of three-independent experiments.

A. Log₂-gene expression ratios normalized to the mean expression in NB samples are shown for diffuse astrocytoma WHO grade II (AII), anaplastic astrocytoma WHO grade III (AAIII), secondary glioblastoma WHO grade IV (sGBIV) and primary glioblastoma WHO grade IV (pGBIV). Median RNA expression is indicated by horizontal bars; boxes show the 25th and 75th percentile range, whiskers mark the 5th and 95th percentiles; maximum and minimum values are depicted as horizontal bars.B, C. The specificity of the antibody against Evi was confirmed by siRNA silencing of the target protein. U87MG cells were transfected with three-independent siRNAs to silence Evi. Silencing of gene expression was validated by Western blot and quantified real-time RT-PCR confirming robust downregulation of Evi expression. β-Actin was detected as loading control. PCR-data are expressed as mean ± SD of three-independent experiments (**p < 0.01).D, E. Representative immunohistochemical stainings for Evi on tissue sections of NB and astrocytic gliomas of different WHO grades. (D) NB, Evi-positive vascular smooth muscle cells; (E) NB, Evi-positive ependymal cells.F, G. Evi-positive tumour cells in a diffuse astrocytoma WHO grade II.H, I. Evi-positive tumour cells in a primary glioblastoma WHO grade IV. Scale bar: 100 µm.