Fig 2: DAB2IP suppresses synaptic protein NLGN3 and NRXN3 expression in GBM.A Volcano plot of false discovery rate (-log10FDR) against fold change between DAB2IP-high and DAB2IP-low U87MG cells from RNA sequencing data (HiSeq 2000 platform, Illumina) was summarized. Some DEG including NLGN3 and NRXN3 are highlighted. B Venn diagram illustrates overlapping alternated-genes among U87MG, LN229, and A172 cells after DAB2IP modulation. C DAB2IP target candidate genes (109 genes) overlapped in U87MG, LN229, and A172 were analyzed using Reactome and highly interconnected networks were constructed. D The expression levels of NLGN3 and NRXN3 mRNA in DAB2IP modulated cells were analyzed by real-time PCR. Red and blue bars indicate DAB2IP-high and DAB2IP-low expression cell lines, respectively. Means ± SD; Student’s two tailed t-test, *p < 0.05, **p < 0.01. E NLGN3 and NRXN3 protein expressions were compared between DAB2IP-high and –low cells by western blot analysis. F After U251 cells were transfected with increment amount of DAB2IP for 48 h, NLGN3 and NRXN3 mRNA expressions were analyzed by real-time PCR. Means ± SD; Student’s two tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001. G Intracellular localization of NLGN3 and NRXN3 proteins were determined by immunocytochemistry, and expression patterns were compared in DAB2IP-high and -low cells. Scale bar=10 µm. Mean fluorescent intensity of NLGN3 or NRXN3 was analyzed using Image J. Means ± SD; Student’s two tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001. H U87MG and LN229 cells were fractionated into cytosol extract (CE), membrane extract (ME), and nuclear extract (NE), then the expressions of NLGN3 and NRXN3 protein were compared in each fraction.

Fig 3: NLGN3 plays a role in maintaining cancer stem cell properties of GBM cells.NLGN3, NRXN3 mRNA (A) and DAB2IP (B) expressions were compared between monolayer (2D) and sphere (3D) culture condition. Red and blue bars indicate DAB2IP-high and DAB2IP-low expression cell lines, respectively. Means ± SD; One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. C After restoration of NLGN3 expressions in U87MG OE (DAB2IP-high, NLGN3-low) and LN229 OE (DAB2IP-high, NLGN3-low) cells, sphere forming abilities including both numbers and size were compared. Red and blue bars indicate DAB2IP-high and DAB2IP-low expression cell lines, green bar indicates NLGN3 restoration in DAB2IP-high cells, respectively. Means ± SD; One-way ANOVA, ***p < 0.001. Scale bar=100 µm. D After restoration of NLGN3 expressions in U87MG OE and LN229 OE cells, CD133 mRNA expression was compared. Means ± SD; One-way ANOVA, *p < 0.05, ***p < 0.001. E After restoration of NLGN3 expressions in U87MG OE and LN229 OE cells, cells were stained with PE-conjugated CD133 and analyzed by flow cytometry. F U87MG OE and LN229 OE cells were co-transfected with NLGN3 and increment amount of NRXN3, and CD133 mRNA expression was compared. Means ± SD; One-way ANOVA, **p < 0.01, ***p < 0.001.

Fig 4: Secreted NLGN3 plays a role in maintaining cancer stem cell properties of GBM cells.A Scheme of NLGN3 functional domains. B NLGN3 and NRXN3 protein expressions were determined in cell lysates and CM derived from DAB2IP-high and –low cells. Primary NLGN3 antibody (NovusBio) detecting N-terminal epitope of NLGN3 was used. C NLGN3 and NRXN3 protein expressions were determined in CM derived from DAB2IP-high and –low cells. Primary NLGN3 antibody (Abcam) detecting C-terminal epitope of NLGN3 was used. D Cells treated with recombinant NLGN3 for 48 h were stained with PE-conjugated CD133 and analyzed by flow cytometry. PE-IgG was used as the negative control for gating and the labels indicated the percentage of each cell population. E U87MG OE and LN229 OE cells were cultured in ultra-low attachment plate under sphere forming culture condition for 14 days, and CM media derived from U87MG Vc and LN229 Vc cells or recombinant NLGN3 protein were added into sphere every 3 days. Means ± SD; One-way ANOVA, **p < 0.01.

Fig 5: DAB2IP regulates NLGN3 transcription through Wnt/ß-catenin signaling pathway.A Scheme of DAB2IP functional domains. B Cells were transfected with various functional domains of DAB2IP and NLGN3 mRNA expressions were determined. Means ± SD; One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. C Cells were transfected with various functional domains of DAB2IP, and cultured in ultra-low attachment plate under sphere forming condition. Sphere forming ability was calculated based on seeding cell numbers. Means ± SD; One-way ANOVA, ***p < 0.001. D Cells were treated with Wnt/ß-catenin inhibitor (LGK974), and NLGN3 protein expressions were compared. E DAB2IP-low cells (A172 KD, U87MG Vc, and LN229 Vc) were treated with LGK974 and NLGN3 mRNA expression was compared. Means ± SD; One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. F DAB2IP modulated cells were transfected with NLGN3 promoter containing luciferase and reporter activity was compared by dual luciferase assay. Means ± SD; One-way ANOVA, **p < 0.01. G DAB2IP-low cells transfected with NLGN3 promoter were treated with LGK974 and reporter activity was determined by dual luciferase assay. Means ± SD; One-way ANOVA, **p < 0.01. H DAB2IP-high cells transfected with NLGN3 promoter were treated with Wnt/ß-catenin agonist, LiCl and reporter activity was determined by dual luciferase assay. Means ± SD; One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.