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Fig. 3 | Molecular Autism

Fig. 3

From: Impaired neurogenesis and neural progenitor fate choice in a human stem cell model of SETBP1 disorder

Fig. 3

SETBP1 deficiency enhances cortical progenitor proliferation. A Cultures were immunostained for dorsal forebrain marker PAX6 (green) at days 30, 40 and 50. Dapi was used to label all nuclei. Scale bar: 100uM. B Quantitative data for PAX6 positive cells presented as mean ± s.e.m for each genotype with a minimum of two independent experiments carried out per line (WT = 5, HET2 = 2, KO1 = 3, and KO2 = 2). One-way ANOVA test with Bonferroni Post Hoc was used to compare the expression between the lines day 30 P = 0.181, day 40 P = 0.032 (Post Hoc WT vs. SETBP1-/-, p = 0.039; WT vs. SETBP1+/-, p = 0.289; SETBP1+/- vs. -/-, p = 0.385), day 50 P = 0.041 (Post Hoc WT vs. SETBP1-/-, p = 0.182; WT vs. SETBP1+/-, p = 0.050; SETBP1+/- vs. -/-, p = 0.473). C WT, SETBP1+/- (HET1) and SETBP1-/- (Homo1) day 35 cultures were immunostained for EdU (green), Ki67 (red), PH3 (red) and counterstained with DAPI (blue). Scale bar: 100uM. D Percentage of cells positive for EdU P = 4.87E−06 (Post Hoc WT vs. SETBP1-/-, p = 1.25E−05; WT vs. SETBP1+/- , p = 0.240; SETBP1+/- vs. -/-, p = 9.01E−06), Ki67 P = 0.016 (Post Hoc WT vs. SETBP1-/-, p = 0.038 WT vs. SETBP1+/- , p = 1; SETBP1+/- vs. -/-, p = 0.025), and EdU and Ki67 (cell cycle re-entry) P = 7.07E−06 (Post Hoc WT vs. SETBP1-/-, p = 1.68E−05 WT vs. SETBP1+/- , p = 0.354; SETBP1+/- vs. -/-, p = 1.38E−05). E Ratios of cell cycle exit and cell cycle length. Data presented as mean ± s.e.m from 3 independent wells with 6 random fields each. One-way ANOVA test (P = 0.055, P = 0.438). F Analysis of PAX6, TBR2 and FAM107A cycling progenitors in Ki67+ population. One-way ANOVA test with Bonferroni Post Hoc, PAX6+ P = 0.00097 (Post Hoc WT vs. SETBP1-/-, p = 0.001; WT vs. SETBP1+/- , p = 0.002; SETBP1 +/- vs. SETBP1-/-, p = 1), FAM107A+ P = 0.141, TBR2.+ P = 0.007 (Post Hoc WT vs. SETBP1-/-, p = 0.036; WT vs. SETBP1+/- , p = 0.996; SETBP1+/- vs. -/-, p = 0.01). G Cell cycle analysis by DNA content using Flow cytometry, % of cells in each of the cell cycle phases. Data presented as mean ± s.e.m of 2 independent experiments in triplicates. Student’s T test, one tail (G1-G0 P = 0.118, S P = 0.255, G2-M P = 0.029). H Growth curve analysis from day 19 to day 45 showing the increased population growth of the SETBP1-/- (Homo1) NPCs compared to the isogenic controls. Statistical significant differences were found from day 30 onwards (Student’s T test, P = 6.71E−05 for day 30, 0.015 for day35, 0.033 for day 40, and 0.050 for day 45). Data presented as mean ± s.e.m from 3 independent wells with two technical measurements. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

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