Figure 4

ID2 restrains differentiation induced by dephosphorylated ASCL1. (A) Relative mRNA expression of different members of the ID family at 7 days after WT and 5S-A ASCL1 expression. Data: mean ± SEM, normalized to TBP. n = 3 independent experiments; one-way ANOVA followed by the Bonferroni post-hoc test; ***p < 0.001; **** p < 0.0001. (B) Quantification of cell confluence. Each data point is mean ± SEM n = 3 independent experiments; one-way ANOVA followed by the Bonferroni post-hoc test; *p ≤ 0.05 between ID2^−/− and 5SA-ASCL1; #p ≤ 0.05 between ID2^−/− and ID2^+/+. (C,D) Representative images of unedited control cells, ID2 wild type cells and ID2 homozygous knock-out cells, without (C) or with (D) dox-induced ASCL1 expression at increasing time points, as labelled. Scale bars: 100 μm. (E) Immunostaining for neuronal marker TUBB3 (green) at 14 days of culture in differentiation conditions. Scale bars: 100 μm. (F) Quantification of the % of TUBB3⁺ cells over the total DAPI⁺ cells at day 14. Data: mean ± SEM n = 3 independent experiments; one-way ANOVA followed by the Bonferroni post-hoc test; *p < 0.05. (H) Quantification of the total neurite length per cell. Data: mean ± SEM n = 3 independent experiments; one-way ANOVA followed by the Bonferroni post-hoc test; ****p < 0.0001.