Fig. 5. IRS1 overexpression maintains CGNP proliferation in the absence of SHH. CGNPs were treated with SHH for 24 hours prior to infection with retrovirus expressing IRS1. (A) Western blotting shows that IRS1 overexpression maintains increased levels of cyclin D2 after SHH withdrawal. (B) RT-PCR analysis shows no effect of IRS1 overexpression on GLI1 or GLI2 expression, but did show an increase in N-myc expression. (C) Levels of CGNP proliferation in response to IRS1-expressing retrovirus infection were assessed with BrdU incorporation. Numbers of BrdU-positive cells are increased with treatment of SHH, as well as treatment with IRS1 retrovirus. Quantification of BrdU incorporation was performed as described in Fig. 4D. (D) To assess effects of virus treatment on cell death CGNPs were stained with antibodies to activated caspase 3. Levels of cleaved caspase 3 remain the same for all treatment groups. Cleaved caspase 3-positive cells were counted and values are expressed as percent of total cells per field. Error bars represent s.e.m., n=5. Asterisks indicate statistically significant difference to untreated CGNPs (P<0.05, n=4).