Materials and Methods

Animal studies
Harvest of cerebellar granule neuron precursors from neonatal mice, preparation of cerebella and tumor tissue from wild-type and mutant mice for histological analysis, and irradiation of tumor-bearing mice were carried out in compliance with the Memorial Sloan-Kettering Institutional animal care and use committee guidelines. NeuroD2-SmoA1 mice were provided by Jim Olson (Fred Hutchinson Cancer Research Center). Patched+/− mice were provided by Kathryn Anderson (Memorial Sloan-Kettering Cancer Center). NOD/SCID mice were purchased from The Jackson Laboratory.

Human tumor collection and expression analysis
Exon array profiling and data analysis with tumor sub-grouping were performed as published (Northcott et al 2009a).

CGNPs and medulloblastoma cell culture
CGNP cultures were generated as previously described (Kenney and Rowitch 2000). Cells were plated on individual poly-DL-ornithine (Sigma) pre-coated plates or pre-coated glass coverslips. In all cases cells were treated with 3 μg/mL of Shh. Where indicated, LY 294002 (Sigma) was used at a concentration of 10 nM for 6h.
Medulloblastoma cells (MBC) were obtained from SmoA1 mouse medulloblastomas. Briefly, tumors were disassociated and cells were incubated in Trypsin/EDTA solution for 30 minutes, then passed through a cell strainer. Cells were subsequently separated on a density step gradient of 35% and 60% Percoll solution (Sigma). Purified MBCs were enriched by pre-plating on uncoated tissue culture dishes to remove adherent fibroblasts and glial cells. Non-adherent cells were plated on tissue culture dishes pre-coated with poly D-lysine (Sigma) and Matrigel (BD Biosciences), infected with retroviruses 24 hours later, and cultured for 24 more hours before implantation or irradiation.

Tumor cell implantation
0.2 million MBC were injected in the cortices of P2 NOD/SCID or C57BL/6 pups with a stereotaxic syringe.

Retrovirus production and infection
The YAP cassette was cloned from pBabe-YAP1 (Addgene) into the retroviral vector pWzl. Short hairpin RNA interference sequences targeting IGF2 at the 3′ or 5′ end of the coding sequence were designed using the Cold Spring Harbor Laboratory RNAi OligoRetriever database and ligated into pSHAG vector, then transferred to MSCV retroviral vector and produced as described (Nahle et al 2008). A control scrambled shRNA retrovirus was also prepared. 293 EBNA (Invitrogen) packaging cells were co-transfected with gag-pol and VSVg packaging plasmids plus pWzl-eGFP, pWzl-YAP1, pMSCV-IGF2shRNA or a control scrambled shRNA using Fugene 6 transfection reagent (Roche). The media was changed 12 hours after transfection and supernatants (8 mL) were harvested at 24 and 48 hours and filtered through 0.45 μm syringe filters. For infection, Shh-treated CGNPs were exposed to the viral supernatants for 3 hours. Viral supernatant was then removed and replaced with fresh medium + Shh.

RNA extraction and Real Time PCR
Total RNA from cerebellar granule neural precursors was extracted and purified using the MiRvana kit (Ambion). cDNA was prepared from 1 μg total RNA by using iScript cDNA Synthesis kit (Bio-Rad). qPCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems). RNA expression data were acquired and analysed using an StepOnePlus Real Time PCR system (Applied Biosystems). Average results and standard errors are presented. TaqMan probes used for qPCR were IGF2 (Mm01163433-m1) and control HPRT1 (Mm01545399-m1).

Protein preparation and immunoblotting
Protein extracts were prepared as previously described (Kenney and Rowitch, 2000). Protein content was determined by using the Bio-Rad protein assay. 50-75 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8-10% polyacrylamide gels and then transferred in 20% methanol buffer at 4°C to Immobilon polyvinylidene difluoride (Millipore) membranes. Immunoblotting was carried out according to standard methods. Antibodies used for western blotting were: YAP1 (Abcam), phospho-Akt (Ser473), phospho-Cdk1(Tyr15), phospho-Chk2 (Thr68), total Akt and cleaved caspase 3 (Cell Signaling), phospho-ATM (Ser1981) (Rockland), Chk2 (Millipore), IGF2 and β-tubulin (Sigma), VEGF, Cyclin D2 and Cyclin B1 (Santa Cruz). Donkey anti-mouse HRP-linked secondary was from Jackson Research Laboratories and goat anti-rabbit from Thermo Scientific. Peroxidase activity was detected using Amershams’s ECL reagents and exposing membranes to Kodak Biomax film.

Cell and tissue immunostaining
For immunofluorescence 8μm sections of paraformaldehyde-fixed/paraffin-embedded tissues were first de-waxed and re-hydrated prior to antigen retrieval. CGNPs were cultured on poly-DL-ornithine coated glass coverslips as previously described. Cells were fixed with 4% paraformaldehyde for 20 minutes. Immunofluorescent staining of sections and cells was carried out according to standard methods. Immunohistochemical staining was performed using a Discovery XT automated staining processor (Ventana Medical Systems, Inc.). Antibodies used were: Ki67 (Vector Labs), YAP1 (Abcam), CD31 (BD Transduction Laboratories), GFP (Invitrogene), BrdU (BD Biosciences), p53BP1 and IGF2 (Novus Biologicals), phospho-histone 3, cleaved caspase 3 and phospho-Akt(Ser473) (Cell Signaling).

Image capturing
Staining of cultured primary cells and tissue sections was visualized with a Leica DM5000B microscope and images were taken using Leica FW400 software. For quantification, TIFF images of 5-10 random fields were taken for each experimental group and average pixel intensities were measured using Image J and Volocity softwares.

Mitosis spreads
Metaphase preparations were done by the Molecular Cytogenetics core facility at Memorial Sloan-Kettering Cancer Center according to standard procedures.

Comet Assay
The comet assay was performed with the Trevigen kit and following the manufacturer’s instructions. Tail lengths were calculated using Comet Assay IV software. Averages from four independent experiments were calculated and representative pictures are shown.

ELISA
Supernatants from CGNPs infected with GFP or YAP and subsequently cultured in 800 μL minimal media without N2 supplement were collected. IGF2 ELISA was performed using the R&D systems kit and following the manufacture’s instructions. Triplicates were used in each case and the average from three independent experiments is shown.