Fig. 4. NICD controls the RNA expression of liver-enriched transcription factors. (A-D) Primary cultures of Dlk⁺ cells were infected with pMX-GFP (GFP), pMX-mutantNICD-GFP (mutant NICD), pMX-NICD-GFP (NICD) and pMX-Hes1-GFP (HES1), kept for 4 days and then used for cDNA synthesis. Quantitative RT-PCR was performed to analyze mRNA levels of HNF1 (A), HNF1ß (B), HNF4 (C), and HNF6 (D) 4 days after infection. Mutant NICD had no effect on the expression of these transcription factors. NICD downregulated HNF1 and HNF4 expression, whereas HNF1ß expression was upregulated and the expression of HNF6 was not significantly affected. Hes1, slightly upregulated the expression of HNF1 and downregulated the one of HNF4. All signals were normalized to the GAPDH levels and to the relative amounts of mRNA (mRNA level in the control culture=1). Analysis was repeated using three sets of samples prepared from three independent cultures. The mRNA levels plotted in the graphs are the average of the three measurements. (E) HNF4 protein expression was examined by immunostaining. Dlk⁺ cells plated in gelatin-coated chamber slides were infected with pMX-GFP (1) or pMX-NICD-GFP (2). After 4 days of incubation, cells were fixed in 4% PFA, permeabilized in MeOH and stained with rabbit anti-mouse HNF4 Ab. Expression of HNF4 was visualized with rhodamine-conjugated anti-rabbit IgG Ab (red). HNF4 expression was downregulated in GFP⁺ cells expressing NICD (green in 1) when compared with the control (2). (F) Expression of C/EBP mRNA. Primary cultures of E14.5 Dlk⁺ cells were infected with viral vectors encoding GFP, Hes1, mutant NICD or NICD. The RNA expression of C/EBP was examined 4 days after infection by northern blot (upper panel). GAPDH expression was also examined as internal control (middle panel). The lower graph shows the level of C/EBP normalized to the GAPDH level, indicating NICD reduced the C/EBP mRNA level to about 60% of that in the control culture (GFP), whereas mutant NICD and Hes1 had no effect. The signals of C/EBP and GAPDH were quantified using the NIH image program.