Specific up-regulation of Hk2 in Shh-treated CGNPs and medulloblastoma. (A) Western blot from isolated CGNPs demonstrates that exogenous Shh induces Hk2 and Cyclin D2. Hk1 expression decreased slightly with exposure to Shh. (B) Colorimetric assay of total Hk activity of CGNP lysates demonstrates a statistically significant increase in Hk activity in Shh-treated CGNPs. (C) Western blot from whole cerebella at indicated days from birth demonstrates temporal expression patterns of Hk1, Hk2 and Cyclin D2 expression. Hk2 and Cyclin D2 were strongly detected during the period of CGNP proliferation (postnatal day (P6, P8) and down-regulated by the end of cerebellar neurogenesis P14. Medulloblastoma, like mitotic CGNPs, expressed high levels of Hk2 and Cyclin D2. (D) Quantitative real-time RT-PCR analysis comparing mRNA expression in Shh-treated CGNPs relative to expression in vehicle-treated CGNPs. Shh induced comparable up-regulation of Hk2 and Cyclin D2. Dots indicate measured fold-change in replicate experiments, and bars indicate mean. (E) Immunofluorescence demonstrates reciprocal patterns of Hk1 and Hk2 at P7. CGNs of the IGL expressed Hk1, which was not detected in CGNPs of the EGL. In contrast, Hk2 was detected only in the EGL, the site of CGNPs at P7. In P16 cerebellum, where the EGL region no longer contains CGNPs, Hk2 was not detected. In medulloblastoma-bearing SmoA1 mice, Hk2 expression was widespread throughout the tumor but remained undetectable in the adjacent IGL. All scale bars = 100 μm.