Fig 2: DAB2IP knockdown affected SCL-1 cell viability and phase. OD490 reflects the number of energetic cells. The changes in the absorption rate of the wavelength 490 nm light by the enzyme marker in the KD and control groups were compared over time. (A) Represents a comparison of the absorption of 490 nm light with time in the KD and control groups. (B) Represents a comparison of the time-varying multiple of light absorption at 490 nm between the KD and control groups. DAB2IP, disabled homolog 2-interacting protein; CON, control; NC, negative control group; KD, knockdown of DAB2IP; OD, optical density. ***, P<0.001.

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: 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 5: ATG9B enrichment is involved in TMZ-induced autophagy.a The mRNA expression of ATG9B analyzed by real-time PCR. pLKO-ATG9B plasmid (shATG9B) was transfected to DAB2IP-low A172 KD or LN229 Vc cells to knockdown ATG9B expression. Black and white bars indicate DAB2IP-high and DAB2IP-low cell lines, respectively. b Representative images of western blot against autophagy-related markers (ATG9B, ATG5, SQSTM1, LC3B) and β-actin. c Flow-cytometry analysis for the detection of acidic vesicular organisms. Flow data are the representative image of two biological experiments. d TMZ log–dose–response analysis (IC50). Cell viability was assessed by MTT assay. IC50 was calculated from curves constructed by plotting the cell survival versus TMZ concentration. The values are expressed as the mean of five technical replicates ± SD. e Colony formation assay. ATG9B knockdown cells and their control cells were seeded in 6-well plates at a density of 1000 cells per well. After 10 days of culture, cells were fixed and stained with 4% formaldehyde, 0.05% crystal violet in PBS for 30min. Absorbance at 590nm was measured after dissolving with 10% acetic acid. Black and white bars indicate DAB2IP-high and DAB2IP-low cell lines, respectively. Means ± SD; n = 3 in a, n = 6 in e; Student’s two-tailed t-test, *p < 0.05, ***p < 0.001.