Acta Neuropathol (2012) 123:587–600 593 Fig. 2 Shh-induced mouse medulloblastomas have high levels of glycolytic markers of hexokinase II, pyruvate kinase M2, and glucose transporter-4. a NeuroD2-SmoA1 medulloblastoma (top row) and adjacent non-tumor cerebellum (bottom row) were subjected to immunoﬂuorescence analyses for glycolysis (HKI, HKII, Glut4, and PKM2). Leftmost panel shows hematoxylin and eosin staining (H&E) staining of the tumor and cerebellar molecular layer (mol) and inner granule layer (IGL). Magniﬁcation: 91.25 (leftmost panel), 940 (other images). Bars 16 lM. Results shown are typical for the 5 different tumor and adjoining cerebellum samples used for immuno- ﬂuorescence in this study. b Protein lysates were prepared from NeuroD2-SmoA1 medulloblastoma and normal cerebellum adjacent to the tumor, and then analyzed by western blotting for glycolysis. 40 lg protein/lane was loaded. The data shown were repeated with multiple sets of tumor and cerebellum from different NeuroD2- SmoA1 mice exhibiting tumors. c Western blot analysis of proteins regulating glycolysis in adjacent non-tumor cerebellum or medullo- blastomas from NeuroD2-SmoA1 mice treated with DMSO (-) or cdk inhibitor olomoucine (?). The same protein lysates from Fig. 1c were blotted for glycolytic markers and this is a typical outcome of those blots this, as it does lipogenesis [2]. We established primary cultures of CGNPs from PN 4/5 neonatal mice. These cells are grown in the absence of serum; addition of exogenous Shh to their medium promotes ongoing proliferation, while in its absence the cells leave the cell cycle and differentiate as they would in vivo. We infected Shh-treated CGNPs with lentiviruses carrying a control, scrambled short hair- pin RNA (shRNA) sequence or a shRNA sequence targeting E2F1 for knock-down. As shown in Fig. 3c and as previously reported, these lentiviruses effectively knocked down E2F1, while infection with the control shRNA had no effect on E2F1 levels. In the presence of 123