Figure 7.

N-myc promotes proliferation and prevents differentiation of PNCs. (A) PNCs were infected with either GFP or N-myc retroviruses and then cultured for 48 h in the absence (untreated) or presence of 25 ng/mL bFGF before being pulsed with tritiated thymidine (³H-Td). Although bFGF inhibited proliferation of GFP-infected cells, little inhibition was seen in cells infected with N-myc viruses. (B–D) PNCs were infected with either GFP or N-myc retroviruses and then cultured for 48 h in the absence or presence of 1 μM cyclopamine. Infected (GFP⁺) cells were FACS-sorted and RNA was isolated and analyzed by real-time RT–PCR using primers specific for Gli1 (B), CyclinD1 (C), or Math1 (D); data are representative of three replicate experiments. (E–H) PNCs cultured in PDL-coated chamber slides were infected and treated with cyclopamine as described above. Infected cells were stained with anti-p27 antibodies and imaged using a Leica AxioImager and Metamorph software. (I) Data from experiments in E-H were quantitated by counting the number of p27⁺/GFP⁺ cells (averaged from four fields per well). These data are representative of five individual replicates of the experiment.