Fig. 4. Nmyc activity in cell cycle regulation lies downstream of Smo. Nmyc functions in a cell-autonomous manner. (A) 48 hours after infection and treatment as indicated below the graph (right), CGNPs (85% pure cultures) were harvested and processed for FACS analysis of cell cycle distribution. Percentages of cells in S-phase were normalized to levels in Shh-treated, uninfected/GFP infected samples. Bars represent average normalization from six samples per treatment. Error bars indicate standard error of the mean. ^*P<0.01, in comparison with Shh-treated control CGNPs. A representative autoradiograph (left) shows western blot analysis of Nmyc (N-myc in figure) and cyclin D1 expression in CGNPs infected and treated with Shh/cyclopamine as indicated above the lanes. Tubulin levels demonstrate equivalent loading of the lanes. (B) Proliferation was assessed using BrdU immunostaining of CGNP cultures enriched for granule cells and treated as indicated below the graph. Numbers of BrdU immunopositive cells in Nmyc+GFP or MB2+GFP-infected cells treated with Shh+cyclopamine or cyclopamine alone were normalized to numbers of BrdU-positive cells in GFP-infected, Shh-treated CGNP cultures. Twenty 100-cell fields were counted per treatment, using two separate litters of animals and two coverslips per treatment per litter. Error bars indicate standard error of the mean. ^*P<0.01, in comparison with Shh-treated control CGNPs. (C) Phenotype of proliferating cells infected with Nmyc-carrying retrovirus. BrdU-GFP, BrdU-GFAP and GFAP-GFP immunostaining (as indicated) was carried out on Shh/cyclopamine treated cultures infected with Nmyc-GFP retrovirus. (Left) BrdU incorporation was exclusively associated with GFP expression (yellow pseudocolor indicates overlap). (Middle) BrdU incorporation was not observed in any GFAP-positive cells. (Right) No GFP-positive cells co-labeled with GFAP, indicating that glial cells were not infected. Note that yellow pseudocolor resulted in this image from a glial process underlying several neuronal cells. DAPI staining (bottom panels) shows distribution of cells.

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