Acta Neuropathol (2012) 123:587–600 591 signaling [14, 15] is marked by strikingly high levels of de novo fatty acid synthesis, as determined by staining for neutral lipids (Fig. 1a, top left, Oil Red O staining), com- pared to the adjacent non-tumorigenic cerebellar tissue (bottom left). This exaggerated lipogenesis is associated with high levels of proliferation as indicated by Ki67 immunoﬂuorescence, and increased cdk activity, leading to Rb inactivation and induction of E2F1 (Fig. 1a, b). Indeed, Fig. 1 Shh-induced mouse medulloblastomas have robust lipogene- sis, increased levels of E2F1 and PPARc and cell proliferation. a Triglyceride accumulation was analyzed in a NeuroD2-SmoA1 medulloblastoma using Oil Red O staining; neutral lipids appear as red droplets. NeuroD2-SmoA1 medulloblastoma (top row) and adjacent non-tumorous cerebellum (bottom row) were subjected to immunoﬂuorescence analyses for E2F1, PPARc, and the proliferation marker Ki67. Primary and secondary antibodies were used at concentrations of 1:100. Leftmost panel shows hematoxylin and eosin staining (H&E) staining of the tumor and cerebellar molecular layer (mol) and inner granule layer (IGL). The non-tumorous cerebellum immunoﬂuorescence focuses on the boundary between the IGL and molecular layer demonstrated in the H&E staining. Magniﬁcation: 91.25 (leftmost panel), 940 (other images). Bars 16 lM. Panels demonstrate the typical outcome from 5 different tumor and adjoining cerebellum specimens all parafﬁn embedded, sectioned, and subjected to immunoﬂuorescence for the aforemen- tioned antibodies. Additionally, the Oil red oil represents a typical outcome based on the staining of frozen sections from 5 different specimens. b Protein lysates were prepared from NeuroD2-SmoA1 medulloblastoma and normal cerebellum adjacent to the tumor, and then analyzed by western blotting for PPARc, lipogenic marker (FASN) and proliferation markers (E2F1 and cyclin D2). 40 lg protein/lane was loaded. Data presented are typical for all 5 sets of tumor and cerebellum samples that were studied. c Western blot analysis of proteins regulating proliferation and lipogenesis in adjacent non-tumor cerebellum or medulloblastomas from NeuroD2-SmoA1 mice treated with DMSO (-) or cdk inhibitor olomoucine (?). The results shown represent a typical outcome amongst the control and experimental groups of mice 123