Fig. 5.

Inhibition of Notch in human NES cells promotes neuronal differentiation. (A) The effect of Notch inhibition on neuronal lineage entry is assessed by qRT-PCR analysis of doublecortin (DCX) expression. Neural stem cells were grown under differentiation conditions for 6 days and RNA was prepared. GAPDH was used for normalisation and values are an average of 2 technical replicates. Data shows representative values of fold-change relative to the DMSO control from one of several independent experiments. Notch specific inhibition with antibodies targeting Notch 1, 2 and 3 generated a 2-fold increase while addition of control antibody had minor effect. Inhibition with the γ-secretase inhibitor DAPT led to a 7-fold increased in DCX expression. (B–D) NES cells were differentiated for 7 days treated with DMSO (B), antibody blockade of Notch1–3 (C) and DAPT (D) for 7 days. Cells were then stained with the post-mitotic neuronal marker beta-III-tubulin (Tuj1) which showed that inhibition of Notch increased the amount of Tuj1 + cells. Experiments were done in duplicates and the proportion of Tuj1 positive cells manually quantified.