Acta Neuropathol (2012) 123:587–600 diseases where glucose metabolism is dysregulated, particularly where sonic hedgehog activities are also implicated [21, 22]. Finally, considering the central role of the Rb-E2F complex in survival and proliferation and the widespread Rb pathway mutations found in human cancers, our ﬁndings could assist in better deﬁning the etiology of tumor metabolism, in particular the perplexing phenotype of aerobic glycolysis and the lack of efﬁcient mitochondrial oxidation of glucose despite the ostensible need for ATP in hyperproliferative cells. The requirement for E2F1 in Shh- regulated PPARc dependent-glycolysis, as demonstrated, highlights its engagement in metabolic core ﬂuxes and underscores the critical role for the Rb/E2F complex in metabolic reprogramming of medulloblastomas and their cells-of-origin. Materials and methods Animal studies Wild type C57-BL6 mice and NeuroD2-SmoA1 mice bearing tumors were administered 10 % dimethyl sulfoxide (DMSO, control), olomoucine (Calbiochem) at 6 mg/kg daily or GW9662 (Cayman Chemicals) at 10 mg/kg daily by intraperitoneal (i.p.) injection. Wild-type C57/BL6 mice, NeuroD2-SmoA1 mice, and E2F1-null mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and typically were 4–5 months of age when demonstrating the signs of a tumor (ataxia, etc.). Cell culture Cerebellar granule neural precursor cultures were gener- ated as previously described in detail [19]. The cell culture media consisted of DMEM-F12 (Gibco, Grand Island, NY) and 19 dilutions of N2 supplement (as 1009 from Gibco), Penicillin Streptomycin (1009 from Gibco) and potassium chloride (2.5 M is a 1009 stock). P4 and P5 wild type C57/ BL6 pups were killed and the cerebellum extracted and placed into 19 Hanks buffered saline solution (HBSS) (Gibco) with 6 gm/L of glucose at a pH of 7.4. The HBSS was aspirated under a tissue hood and trypsin (Gibco) was added with 1 mg/ml of DNAse. The trypsin was then inactivated with the media containing 10 % FCS and the cerebella titurated twice. Following a 5 min spin at 1,500 rpm, the pellet was resuspended in 10 % FCS media. Cells were then plated on polyornithine-coated six well plates at a concentration of 3 million cells/mL in 1 mL of the 10 % FCS media for 6–12 h. The media were then replaced with FCS free media and given their treatments and incubated for 24 h before protein lysis. Where indi- cated Shh (R&D Systems, Minneapolis, MN, USA) was 589 used at a concentration of 3 lg/ml, cyclopamine (R&D Systems) was used at 1 lg/ml and GW9662 (Cayman Chemical, Ann Arbor, MI, USA) was used at 50 or 100 nM. Western blotting Protein extracts were prepared as previously described [20]. A total of 40 lg of murine cerebellum, medullo- blastoma, or CGNP protein extract were run on 8–12 % SDS–polyacrylamide gels and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The blots were blocked and incubated with primary antibodies in 5 % milk in Tris-buffered saline-Tween (TBS-T) overnight in 4 C. Blots were washed three times and incubated with sec- ondary antibodies in 5 % milk in TBS-T for 2 h in room temperature. After washing, the signals were developed using the enhanced chemiluminescence method (GE Healthcare, Piscataway, NJ, USA) and the membranes were exposed to Kodak Biomax ﬁlm. Primary antibodies were: E2F1 (Cell Signaling, Danvers, MA, USA), PPARc (E-8; Santa Cruz Biotechnology, Santa Cruz, CA, USA), FASN (Cell Signaling), Hexokinase I (Cell Signaling), Hexokinase II (Cell Signaling), Glut4 (Cell Signaling), PKM2 (Cell Signaling), Cyclin D2 (M-20; Santa Cruz) and b-tubulin (Sigma). Horseradish peroxidase-conjugated secondary antibodies were: goat anti-rabbit IgG (H–L; Thermo Scientiﬁc, Rockford, IL, USA) and donkey anti- mouse IgG (H–L; Jackson Immuno-Research, West Grove, PA, USA). Primary antibodies were diluted to 1:1000 for incubation in 5 % milk in TBS-T with the exception of b-tubulin, which was 1:5,000 while the secondary antibodies were at 1:10,000 as recommended by their manufacturers. Lentivirus production and infection E2F1 shRNA and scrambled control lentivirus constructs were obtained from The RNAi Consortium (Sigma, St Louis MO). Each construct was transfected into the Pzp53med cell line, which was derived from a medullo- blastoma arising in a Ptc?/-/p53-/- mouse [1], and western blotting was used to determine which shRNAs effectively and speciﬁcally targeted E2F1. These constructs were used to prepare lentiviruses. 293T cells grown in 10 % FBS Dulbecco’s modiﬁed Eagle’s medium (Gibco) were transfected with E2F1 or scrambled control shRNA lentiviral vectors and MISSION lentiviral packaging mix (Sigma) using Fugene 6 in serum-free OPTI-MEM medium (Invitrogen, Carlsbad, CA, USA). Lentiviral supernatants were collected 48-h post-transfection and ﬁltered through a 0.45 l ﬁlter, then pooled. For infection, Shh-treated CGNPs were exposed to the lentiviral supernatant for 4 h. Control scrambled shRNA lentivirus constructs were used 123