Fig. 3. Promotion of hepatic differentiation by disrupting Notch signaling. (A) Downregulation of Notch2 gene expression by siRNA. E14.5 Dlk⁺ cells were transfected with siRNA specific for Notch2 mRNA using oligofectamin 18 hours after plating. The mRNA levels of Notch2 and those of TAT and CPS were examined 4 days (Notch2) and 6 days (TAT and CPS) after lipofection by northern blotting. GAPDH expression was also examined as internal control. siRNA reduced mRNA levels of Notch2 by to about 60% of that in the control culture. Hepatic differentiation markers TAT and CPS were upregulated in the presence of siRNA. (B) Inhibition of presenilin. The -secretase inhibitor L685,458 in DMSO was added to the primary culture of E14.5 Dlk⁺ cells. After 7 days of incubation, total RNA was extracted from each well and used for northern blot analysis to detect marker gene expression. Hepatic differentiation markers TAT and CPS were upregulated in the primary culture of Dlk⁺ cells. GAPDH expression was examined as a control. (C,D) Morphological examination of the cultures showed that addition of the presenilin inhibitor induced the formation of clusters, containing cells with condensed cytosol and clear round-shape nuclei (D) when compared with the addition of DMSO (C).