Fig 1: CCN3 promotes proliferation and inhibits the neuronal differentiation of culture mouse hippocampal NSCs. The effect of CCN3 on NSC proliferation was measured by BrdU (A) and Ki-67 (B). Each single data points from nine independent cell culture preparations (n = 9) are presented as the percentage of BrdU+ cells among PI-stained cells (C) or Ki-67+ cells (D) among DAPI-stained cells. (E) Cell cycle analysis was used to detect the proliferation index (PI). The result is expressed as the mean ± SD with single data points from three independent cell culture preparations (n = 3). The number of Tuj1- (F) and DCX- (G) positive cells was determined by immunostaining. Each single data points from nine independent cell culture preparations (n = 9) are presented as the percentage of Tuj1+ (H) and DCX+ cells (I) among DAPI-stained cells. (J,K) Representative WB images illustrated the protein expression of Tuj1, and β-Actin was used as a reference protein. The ratio of Tuj1 to β-Actin was quantified using WB band analysis, and the result is expressed as the mean ± SD with single data points from three independent cell culture preparations (n = 3). Scale bars in (A,B,F,G) denote 50 μm. Differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. The Kolmogorov–Smirnov test was used for normality and homogeneity. * p < 0.05 and ** p < 0.01 versus the Ctrl group; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the shNC group.
Fig 2: CCN3 promotes NSC proliferation in the mouse dental gyrus. (A) Following the same grouping as before, Ki-67 and SOX2 double immunostaining was used to detect NSC proliferation. Quantitative analysis of the number of Ki-67 (B), SOX2 (C), and double-positive cells (D), each data represents the mean ± SD from 9 animals (n = 9). Scale bar = 50 μm. Differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. The Kolmogorov–Smirnov test was used for normality and homogeneity. * p < 0.05, ** p < 0.05, and *** p < 0.001 versus the Ctrl group; # p < 0.05 and ## p < 0.001 versus the shNC group.
Fig 3: Alteration of CCN3 expression has little impact on cell apoptosis. Cells were transfected with either a Ccn3-targeting shRNA (shCCN3) or a non-specific shRNA (shNC). After 7 days, eGFP-positive cells were found in both the DG area (A) and cultured hippocampal NSCs (B). (C,D) WB band quantification for the ratio of CCN3 to β-Actin was presented, and each value represents the mean ± SD of three independent experiments (n = 3). Differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. *** p < 0.001 versus the shNC group. The schematic workflow of the experiment involving the injection of lentivirus (E) and CCN3 protein solution (F) is as follows. Mice were randomly assigned to different treatment groups and received a DG infusion of normal saline solution (Ctrl), CCN3 protein solution (CCN3), lentiviral shRNA against Ccn3 (shCCN3), or control shRNA (shNC). (G–I) TUNEL staining was utilized to detect apoptotic cells. Data are presented as the percentage of TUNEL-positive cells in the total DAPI-stained cells, and each value represents the mean ± SD of nine independent experiments (n = 9). Differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Scale bars in (A,B,G) represent 100 μm; in (H), 50 μm.
Fig 4: Culture of mouse hippocampal NSCs and the effect of CCN3 on cell viability. Neural stem cells (NSCs) were isolated from the hippocampus of neonatal mice. Following 3–5 days of culturing, 90–170 μm neurospheres were observed (A) and the majority of these cells expressed nestin (B). (C) The NSCs were identified using double-immunofluorescent labeling with nestin and SOX2. DAPI was used as a counterstain for the nuclei. (D–F) Tuj1-, GFAP-, or NG2-positive cells were observed following normal differentiation medium culturing for 3 days. (G) CCN3 expression co-localized with nestin, as observed by double immunofluorescent staining. (H) NSCs were exposed to varying concentrations of CCN3 (5, 10, 20, 50, 100, 200, and 500 ng/mL) for 3 days, and cell viability was measured using the CCK-8 assay. All results are expressed as the mean ± SD with single data points from at least three independent experiments. Statistical analysis was performed using one-way ANOVA. The Kolmogorov–Smirnov test was used for normality and homogeneity. * p < 0.05; *** p < 0.001 versus normal control (0 ng/mL). Scale bars in (A,B) denote 100 μm; in (C–G), 50 μm.
Fig 5: Notch/PTEN/AKT pathway is closely involved in regulating the effect of CCN3 in mouse hippocampal NSCs. After the modulation of CCN3 expression, NSCs were treated with a vehicle (DMSO), 20 μM of FLI-06, or 5 μM of VO-OHpic trihydrate (VO-OH). (A,B) BrdU staining and Tuj1 staining were performed to detect proliferation and neuronal differentiation, respectively. Scale bar = 50 μm. Each single data points from nine independent cell culture preparations (n = 9) are presented as the percentage of BrdU+ cells among PI-stained cells (C) or Tuj1+ cells (D) among DAPI-stained cells. Differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. The Kolmogorov–Smirnov test was used for normality and homogeneity. * p < 0.05, ** p < 0.01, and *** p < 0.01 versus the DMSO group; ### p < 0.001 versus the CCN3 group; &&& p < 0.001 versus the shCCN3 group. (E) The illustration presents the mechanisms by which CCN3 regulates proliferation and differentiation in NSCs.
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