Fig 2: Differentiation efficiency of various neuron inducers, identified by immunofluorescence.a Images of immunofluorescence using NeuN, GFAP, Tuj1 and Nestin as characteristic markers of NGF/CNTF/NT4/MT-treated cells at 5D (5 days), 3D (3 days) and 1D (1 day). Quantification of immunostaining data. The y-axis shows the number of: b NeuN- and c GFAP-positive cells, n = 3 imaging field repeats. Data are shown as mean ± SEM. Statistical significance was determined by two-sided Welch’s ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 100 μm.

Fig 3: FOXG1 induces final mature of GNs in adult hippocampus DG and increases synaptic plasticity. (A) Expression patterns of NeuN-expressing GNs in the hippocampal DG of Foxg1 genotype mice were assessed with immunofluorescence. Red = NeuN; Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. Arrow heads: newly generated GNs with FOXG1 activation. (B) Levels of total GNs in adult hippocampus are showed as %NeuN+/DAPI+ cells. (C) Levels of endogenous GNs in adult hippocampus are showed as %GFP−NeuN+/DAPI+ cells. (D) Expression patterns and (E) qualification of total GNs in adult hippocampus were studied with immunohischemisitry. Scale bars = 100 μm. (F,G) Hippocampal lysates of the two genotype mice were immunoblotted using an antibody against NeuN. GAPDH was used as the loading control. Values are expressed as means ± S. E. M. For each group, n = 6. Significant levels set at * p < 0.05, *** p < 0.001 noted difference between Foxg1fl/fl and Foxg1fl/fl-CreAAV animals. (H) Fluorescence microscopic analysis of differentiation-related morphological changes of N2A cells by stained the F-actin cytoskeleton with fluorescently labeled phalloidin. Green = Phalloidin; Blue = DAPI. As positive control, cells were differentiated with 10 μM RA. Scale bars = 25 μm. (I) Quantification of the average neurite lengths are illustrated in the column diagrams. Values are expressed as means ± S. E. M. For each group, n = 6. Different letters indicate statistical differences in mean values among groups (p < 0.05). ns—not significant.

Fig 4: Results of Nissl staining and double immunofluorescence staining for NeuN and Iba-1 in the mouse hippocampus. (A) Representative images and (B and C) quantitative data for the number of Nissl-positive hippocampal neurons. Both 10 and 25 nM/kg semaglutide increased the numbers of Nissl-positive hippocampal neurons in the CA1 and CA3 areas (one-way ANOVA). (D) Representative immunofluorescence images and (E and F) densitometric analysis of NeuN and Iba-1 showing that both doses of semaglutide (enhanced the fluorescence intensity of Neun and decreased the fluorescence intensity of Iba-1 in the CA1 and CA3 areas (two-way ANOVA). Data are presented as the means ± SD. *P<0.05, **P<0.01 and ***P<0.001. N≥3. PTZ, pentylenetetrazole.

Fig 5: Differentiation efficiency of training and testing sets identified by immunofluorescence, western blot and RT-qPCR.a Images of immunofluorescent staining using NeuN, GFAP, Tuj1 and Nestin as characteristic markers of neuron and astrocyte differentiation induction at 5D (5 days), 3D (3 days), 2D (2 days), 1D (1 day) and 0.5D (0.5 days). Differentiated cells treated with NT3 and LDH-NT3 were stained following the same protocols. Quantification of immunostaining data. The y-axis shows the number of: b NeuN- and c GFAP-positive cells, n = 3 imaging field repeats. d Western blot analysis of NeuN, GFAP, Nestin and Tuj1 protein in NSCs in different states of differentiation, n = 3 biological repeats. e Quantitative real-time PCR detection of NeuN, GFAP, Nestin and Tuj1 gene expression in cells in different states of differentiation, n = 3 biological repeats. Data are shown as mean ± SEM. Statistical significance was determined by two-sided Welch’s ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 100 μm. LDH: layered double hydroxide.