594 Acta Neuropathol (2012) 123:587–600 Fig. 3 Loss of E2F1 in mice ablates PPARc, and key markers of de novo lipogenesis, glycolysis, and cell proliferation. a PN 7 sagittal sections of wild-type (bottom row) and E2F1-null (top row) cerebella were immunostained for the glycolysis-related proteins Glut4, muscle pyruvate kinase 2 (PKM2), hexokinase 1 and 2 (HKI; HKII) as well as PPARc, and E2F1 with the proliferation marker phospho-histone H3. First column panel shows cerebellar inner granule layer (IGL), Purkinje cells (PC), and external granule layer (EGL). Magniﬁcation: 940. Bars 32 lM. These slides are indicative of several specimens from the wildtype and E2F groups that were analyzed with IF. Shh, CGNPs have increased levels of PPARc, HKII, PKM2, and Glut4, consistent with our observations in vivo. Levels of HKI were unaffected by the presence of Shh. Infection with the scrambled shRNA results in a slight reduction of PPARc levels but has no effect on levels of the glycolytic markers. In contrast, E2F1 knock-down effec- tively abrogated expression of PPARc and the associated glycolytic markers. Taken together, the results of our in vivo analysis and in vitro manipulation of E2F1 indicate that in Shh-associated mouse medulloblastomas and in proliferating CGNPs, glycolysis is elevated in an E2F1- dependent manner and its regulation functionally lie downstream of PPARc. 123 b Cerebella were collected from wild-type or E2F1-null PN 5 pups, then analyzed by western blotting for levels of E2F1, PPARc, glycolysis proteins (HKI, HKII, PKM2, and Glut4), the lipogenic marker fatty acid synthase (FASN), and proliferation marker cyclin D2. Results are typical of several sets of protein lysates that were collected. c Western blot analysis of E2F1, PPARc, glycolysis (HKI, HKII, PKM2, Glut4) and proliferation marker cyclin D2 in CGNPs treated with DMSO vehicle, Shh or Shh in the presence of lentiviruses carrying shRNAs targeting E2F1 or a scrambled control shRNA virus PPARc activity is required for the expression of glycolytic markers in CGNPs CGNPs require Shh pathway activity for proliferation as well as for E2F1 expression. To establish that PPARc and glycolysis are regulated by Shh upstream of E2F1, we cultured CGNPs in the presence of Shh or in the presence of Shh and cyclopamine, which inhibits Smoothened and blocks Shh signaling. As we have shown previously, cyclopamine treatment blunts the accumulation of E2F1 and FASN, and reduced proliferation as determined by cyclin D2 levels (Fig. 4a). Exposure to cyclopamine had no effect on HKI. However, in the presence of cyclopamine