Fig 2: High levels of NLGN3 cause glioma cells to grow faster. (A‐G) Cell count analysis of U251 cells in 0 h (A), 12 h (B), 24 h (C), 36 h (D), 48 h (E), 60 h (F), and 72 h (G) after treatment with different concentration of NLGN3 (0, 10, 20, 30, 40, 50 nmol/L) (n = 6 independent cultures). (H‐N) Cell count analysis of U87‐MG cells in 0 h (H), 8 h (I), 16 h (J), 24 h (K), 32 h (L), 40 h (M), and 48 h (N) after treatment with different concentration of NLGN3 (0, 10, 20, 30, 40, 50 nM) (n = 6 independent cultures). (O and P) Western blots analysis of p‐AKT (S473) level (O) in U251 cells 48 h after treated with NLGN3 (0, 10, 20, 30, 40, 50 nmol/L); quantification analysis of p‐AKT level (P) shows an increased expression of normalized p‐AKT compared with 0 nM group (n = 6 independent cultures, *P < .05 vs the 0 nmol/L). (Q and R) Western blots analysis of p‐AKT (S473) level (Q) in U87‐MG cells 32 h after treated with NLGN3 (0, 10, 20, 30, 40, 50 nmol/L); quantification analysis of p‐AKT level (R) shows an increased expression of normalized p‐AKT compared with 0 nmol/L group (n = 6 independent cultures, *P < .05 vs the 0 nmol/L). The data are expressed as mean ± SE. Statistical analysis was implemented by student's t test and variance analysis

Fig 3: NLGN3 in the deep brain regions is higher than in the cortex in the pathological conditions of GBM. (A and B) Diagram shows the dissected structural domain of frontal lobe, temporal lobe, parietal lobe, occipital lobe, basal ganglia, thalamus, and corpus callosum in normal human brain. (C and D) Western blotting analysis of NLGN3 levels in the normal human brain frontal lobe, temporal lobe, parietal lobe, occipital lobe, basal ganglia, thalamus, and corpus callosum (C), quantification analysis of NLGN3 levels (D) shows a higher NLGN3 levels in the cortex (n = 5, *P < .05 vs the cortex). (E) Schematic diagram showing the dissected structural domain of cortex and core in normal mouse brain. (F and G) Western blotting analysis of NLGN3 levels in the normal mouse brain cortex and core (F), quantification analysis of NLGN3 levels (G) shows a higher NLGN3 levels in the cortex (n = 6, *P < .05 vs the cortex). (H) Schematic illustration showing the surface and fundus structural domain of GBM tissue from patients, the normal cortex and normal deep brain regions anatomical schematic in the GBM patients brain. (I‐L) Western blotting analysis of NLGN3 (I) and p‐AKT(S473) (K) levels in the surface and fundus of GBM tissues, quantification analysis of NLGN3 (J) and p‐AKT(S473) (L) levels shows a higher NLGN3 and p‐AKT(S473) levels in the fundus region (n = 3, *P < .05 vs the surface region). (M‐P) Western blots showing the NLGN3 (M) and p‐AKT(S473) (O) levels in the normal cortex and normal deep brain regions from GBM patients after surgery, quantification analysis of NLGN3 (N) and p‐AKT(S473) (P) levels shows a higher NLGN3 levels in the normal deep brain region (n = 3, *P < .05 vs the normal cortex). The data are expressed as mean ± SE. Statistical analysis was implemented by student's t test and variance analysis

Fig 4: NLGN3 in the deep brain regions is lower than in the cortex in the normal human brain. (A and B) Immunofluorescence staining analysis of NLGN3 in the normal human brain frontal lobe, temporal lobe, parietal lobe, and occipital lobe (A); basal ganglia, thalamus, and corpus callosum (B). Scale bar, 20 µm. (C) The optical density (OD) analysis of NLGN3 expression (n = 5, *P < .05 vs the cortex). The data are expressed as mean ± SE. Statistical analysis was implemented by student's t test and variance analysis

Fig 5: NLGN3 in the deep brain regions is higher than in the cortex under the GBM pathological circumstances. (A and B) Immunofluorescence staining analysis of NLGN3 in normal mouse brain cortex and core (A), the optical density (OD) analysis of NLGN3 expression (B) (n = 6, *P < .05 vs the cortex). Scale bar, 20 µm. (C and D) Immunofluorescence staining of NLGN3 in the two stage of GBM tissues (C), the optical density (OD) of NLGN3 expression (D) (n = 3, *P < .05 vs the surface region). Scale bar, 20 µm. The data are expressed as mean ± SE. Statistical analysis was implemented by student's t test and variance analysis