C/EBPα/β and NeuroD transcription factors are co-expressed in neurons and cooperate to activate the Fos promoter. (a,b) Immunocytochemical analysis of cortical neuron progenitors cultured at E12.5, and stained with Nestin antibodies after 2DIV (a) or antibodies against the differentiation marker MAP2 after 4DIV (b). (c) RT-PCR analysis of Cebpa/b and Neurod in cortical progenitors. Primary cortical neuron progenitors from two different preparations were cultured at E12.5 and harvested for total mRNA after 2DIV (lanes 1 and 2), 4DIV (lanes 3 and 4), or 6DIV (lanes 5 and 6). Ubiquitin was used to control the levels of mRNA. Lanes 7 and 8 are mRNA samples without reverse transcriptase (RT). Lane 9, no template. (d) RT-PCR shown in (c) was quantified by densitometric analysis and is shown as the ratio of transcription factor (TF) to ubiquitin expression level. (e) Cooperation between C/EBPα and NeuroD transcription factors in the activation of the Fos promoter. NIH3T3 cells were transfected with either a wild-type Fos reporter plasmid (pWT, blue bars) or a reporter plasmid in which both the C/EBP and the Ebox binding sites were mutated (pMcebp/ebox, red bars), in the absence (lanes 1 and 5) or presence of a plasmid containing either C/EBPα (lanes 2 and 6), NeuroD (lanes 3 and 7) or a combination of both plasmids C/EBPα and NeuroD (lanes 4 and 8). Co-transfection of C/EBPα and NeuroD significantly increased the activation of the wild-type Fos promoter (lane 4) compared to C/EBPα or NeuroD alone (p < 0.0001 in both cases). This cooperation was significantly reduced (p < 0.0001) upon mutation of the C/EBP and Ebox binding sites (lane 8). Luciferase activity was normalised to β-gal activity. The relative luciferase activity represents the mean value of at least four independent experiments.