Fig. 4. Ngn2 and E47 can rescue NICD induced block of neurogenesis. (A-C,T) Cells transfected with NICD (0.7 µg/µl) for 42 hours were Sox3⁺ (A) and incorporated BrdU (B), but had failed to upregulate the expression of the neuronal markers NeuN and Tuj1 (C,T). (D-I,T) Co-transfection with either Ngn2 or E47 (0.7 µg/µl) did not block the capacity of NICD to maintain cells in an undifferentiated and self-renewing state. (J-L,T) Forty-two hours after electroporation, neural cells co-transfected with Ngn2, E47 and NICD (0.7 µg/µl of each expression vector) had downregulated Sox3 (J), exited the cell cycle (K) and upregulated the expression of neuronal markers (L,T). (M) The Notch responsive reporter construct, 12xCSL-DsRed, was highly activated in NICD-transfected cells. (N,O) These cells were maintained in a Sox3-expressing state (N) and failed to upregulate the expression of Tuj1 (O). (P-R) In cells co-transfected with NICD/NGN2/E47 the 12xCSL-DsRed reporter construct was expressed in cells located in the marginal zone (P) that had downregulated Sox3 (Q) and instead upregulated the expression of Tuj1 (R). (S) Misexpression of Sox3 efficiently blocked the generation of neurons, even when co-transfected with high levels of Ngn2 and E47. (T) Quantification of the number of electroporated cells expressing the neuronal marker NeuN. The white square in A indicates regions analyzed. The figures in T are represented as percentage of electroporated cells expressing NeuN, mean±s.e.m. ^**P<0.01, ^***P<0.001, relative to EGFP-transfected control cells, Student's t-test. Scale bar: 40 µm in P; 10 µm in S.