Figure 1

ASCL1 phosphorylation regulates proliferation of human GSCs. (A) ASCL1 protein expression in GBM cell lines; positive control is after ASCL1 overexpression. (B) Western blot showing endogenous ASCL1 phosphorylation in GBM cells, with and without phosphatase (λ-PP) treatment. (C) Overexpression of WT and 5S-A ASCL1 in G144 cells, with and without λ-phosphatase treatment. White arrowheads in B and C indicate phosphorylated ASCL1, while black arrowheads indicate unphosphorylated ASCL1. (D) Representative images of G144 cells after growth factor withdrawal and dox-induced WT and 5S-A ASCL1 expression. Scale bars: 300 μm. (E) 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; ***p < 0.001. All data points from day 3 onwards statistically differ between control and WT ASCL1 and between control and 5S-A ASCL1 (p < 0.05; not shown). (F) Quantification of cell number at 2, 7 and 14 days of ASCL1 induction. Data: mean ± SEM n = 3 independent experiments, t-test; **p < 0.01; ****p < 0.0001 (G) Immunofluorescence showing EdU incorporation. Scale bars: 100 μm. (H) Quantification of the % of EdU-positive cells. Data: mean ± SEM n = 3 independent experiments, t-test; *p < 0.05. (I) Expression of negative cell cycle regulators in G144 cells after 7 days of ASCL1 induction. Data: mean ± SEM, normalized to TBP. n = 3 independent experiments; one-way ANOVA followed by the Bonferroni post-hoc test; **p < 0.01; ***p < 0.001. Full length western blots are provided in Supplementary Fig. S1A, B and C.