Additional file 1 of Decreased nuclear Pten in neural stem cells contributes to deficits in neuronal maturation
Creators: name: Kang, Shin Chung
name: Ritika Jaini
name: Hitomi, Masahiro
name: Hyunpil Lee
name: Sarn, Nick
name: Stetson Thacker
name: Eng, Charis
Publisher: name: figshare
Year: 2020
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Developmental Biology
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 1: Supplementary Information. Decreased nuclear Pten in neural stem cells contributes to deficits in neuronal maturation. Figure S1. Neural stem cells (NSCs) derived from Ptenm3m4 mice have higher stemness characteristics. a NSCs derived from dentate gyrus (DG) of wildtype (left, n=3), Ptenwt/m3m4 (center, n=4), and Ptenm3m4/m3m4 (right, n=4) mice had similar morphology after 1 day and 5 days (n=3) of in vitro growth in cell cultures containing growth factors (EGF and FGF2). Images shown in 20X. Ptenm3m4 mutant NSCs showed a lack of contact inhibition (white arrow). b Quantification of c-Myc staining in Figure 1a, showing a significant increase in c-Myc expression in the homozygous mutant relative to wildtype (p-value = 0.051). c Quantification of c-Myc expression at 5 DIV finding a significant increase in expression for Ptenm3m4/m3m4 NSCs compared to wildtype (p-value = 0.035) and Ptenwt/m3m4 (p-value = 0.0042) NSCs (p-value = 0.035) as assessed by one-way ANOVA with Tukey-Kramer post hoc testing. d Quantification of Ki67 immunofluorescence at 0 and 3 DIV, finding a significant difference in Ki67+ nuclei between Ptenm3m4/m3m4 and wildtype NSC at 0 (p-value = 0.036) and 3 (p-value &lt; 0.0001) DIV as assessed by one-way ANOVA with Tukey-Kramer post hoc testing. There was also a significant increase in Ki67+ nuclei between Ptenwt/m3m4 and wildtype NSCs at 3 DIV (p-value = 0.0039) as assessed by one-way ANOVA with Tukey-Kramer post hoc testing e Western blot analysis of undifferentiated NSCs (n = 3), showing increased Ccnd1 (p-value = 0.014) and P27kip1 (p-value = 0.039) expression in Ptenm3m4/m3m4 NSCs compared to wildtype NSCs as assessed by ANOVA with Tukey-Kramer post hoc testing. A significant difference in expression of Ccnd1 between Ptenm3m4/m3m4 and Ptenwt/m3m4 NSCs (p-value = 0.011) was also found as assessed by ANOVA with Tukey-Kramer post hoc testing (*p-value &lt; 0.05; **p-value &lt; 0.01; ****p-value &lt; 0.0001). Figure S2. NSCs with Ptenm3m4 mutations show decreased nuclear and global Pten levels. a Densitometry quantification of ratio of Pten expression normalized to Gapdh in Ptenm3m4/m3m4 NSCs vs Ptenwt/wt NSCs at 1 DIV (p-value = 0.0042). b Densitometric quantification of Western blot analyses of Pten expression normalized to Gapdh over 5 days of random differentiation. Two-way ANOVA testing found differences in Pten expression (p-value = 0.021) and between the time points (p-value = 0.036), where Pten expression explains 38% of the variance and time in culture explains 15%. No interaction was found between factors (Pten expression and DIV) was found. c Densitometric quantification of Western blot analysis of Pten, p110α (Pi3k), p-mTor, and p-Akt expression in Ptenm3m4 NSCs. Kruskal-Wallis testing, accompanied by Dunn’s multiple comparison testing, found a significant decrease in Pten (p-value = 0.034) and a significant increase in p-mTOR (p-value = 0.034) and p-Akt (p-value = 0.034) in Ptenm3m4/m3m4 versus wildtype NSCs. Figure S3. Synapthophysin staining on Ptenm3m4 NSCs. a High magnification representative image of Syn+ (green) cells at seven DIV with DAPI (blue). Scale bar = 10 μm. b Top panel: Low magnification representative image of Syn+ (green) cells at 10 DIV with DAPI (blue). Bottom panel: High magnification of circled image in top panel. Scale bar = 20 μm. c Quantification percent Syn+ cells normalized to DAPI expression. Only significant difference between wildtype Syn expression at seven and 10 DIV (p-value &lt; 0.05). Scale bar = 20 μm. Figure S4. Quantitative view of gliagenesis in Ptenm3m4 NSCs. a Quantification of gliagenesis staining data in Figure 5, including Ng2, Olig1, Mbp, and Gfap. Gfap shows a significant difference in expression between wildtype and homozygous mutant by integrated density or stain area normalized to cell number (p-value = 0.032 and 0.017, respectively). b Gfap staining trace analysis followed by stain area and perimeter calculations.