Fig. 2
ASCL1 overexpression in GBM CSCs promotes neuronal differentiation and lineage switch. a ASCL1 overexpression in CSCs significantly reduces their long-term self-renewal (population analysis; representative analysis of n = 3 experiments; p value of the comparison EGFRpos mock vs. ASCL1-overexpressing CSCs < 0.05; p value of the comparison EGFRneg mock vs. ASCL1-overexpressing CSCs < 0.05). b ERK and AKT pathways are hypoactivated in ASCL1-transduced GBM CSCs (WB). c ASCL1 overexpression in CSCs induces neuronal differentiation under proliferative (upper panels) and differentiative (lower panels) conditions, as assessed by early (Tuj1) and late (MAP2) neuronal markers (Tuj1, red, ×400; MAP2, red, inset ×400). d ASCL1-induced neuronal differentiation of CSCs is accompanied by the concurrent repression of the astroglial phenotype under both proliferative (upper left panels) and differentiative (lower left panels) conditions (Tuj1, red; GFAP, green; ×400). Quantification of the frequency of Tuj1- and GFAP-IR cells after ASCL1 overexpression (right panels). e ASCL1 overexpression induces CSCs to invade as clusters of cells (black arrows), whereas mock CSCs move as single cells (left panels) (standard Matrigel Transwell invasion assay). ASCL1 overexpression increases invasion (30 min), triggers a shift from single-cell to collective migration (1 h; representative CSC line L0512), and induces the formation of heterogeneous ‘niche’-like structures (3–16 h; epithelioid cells: black arrows; ‘satellite cells’: white arrows; the pseudocolor magenta identifies the Matrigel layer; representative CSC line L0605) (scanning electron microscopy, SEM, Matrigel Transwell invasion assay, right panels)
