Supplementary Figure S1 Nestin expression in Hh type MB
A-B, MB cells isolated from Math1-Cre/R26R-smoM2 mice (A), or ptch1 heterozygous mice (B, ptch1lacZ mice) were immunostained for Nestin and counterstained with DAPI. C-D, Sagittal sections from a
Nestin-CFP/Math1-Cre/ptch1C/C cerebellum at P7, were immunostained for CFP and S100 (C) or CFP and
Zic1 (D). CFP+ cells in both molecular layer (ML, arrows) and internal granule layer (IGL, arrow heads)
were S100+ (C) and Zic1- (D), suggesting that Bergmann glial cells, not granule neurons express Nestin in
the ML and IGL. E, Two different primer sets were designed to analyze ptch1 genomic DNA. One primer
set amplifies part of exon 3, which is deleted in the mutant allele. The other primer set is specific for exon
14, which is present in both wildtype and mutant alleles. The amount of undeleted ptch1 in each sample is
reflected the level of exon 3 product. The total amount of ptch1 DNA is represented by exon 14 product.
Comparable levels of ptch1 deletion were detected in Nestin+ and Nestin- GNPs. Brain endothelial cells
(bEnd.3 cell line from ATCC) were used as a control. F, mRNA expression of gli1 and n-myc in wildtype
GNPs, Nestin+ and Nestin- ptch1-deficient GNPs was examined by qRT-PCR.
Supplementary Figure S2 ptch1-deficient GNPs develop MB upon transplantation
Wholemount brain images of CB17/SCID mice after transplantation with CFP+ cells (A) and CFP- cells
(B) purified from Nestin-CFP/Math1-Cre/ptch1C/C mice. Arrows point to tumors.
Supplementary Figure S3 gli3 mRNA expression and cell cycle distribution in NIH3T3 cells and
ptch1-deficient GNPs
a-c, The expression of gli3 mRNA in Nestin+ and Nestin- GNPs from Nestin-CFP/Math1-Cre/ptch1C/C
mice at P7 (A), in NIH3T3 cells transfected with GFP (WT), Nestin shRNA (Nes shRNA) or Nestin (Nes
O/E) after treatment with DMSO (B) or 3μg/ml recombinant Shh (C), was determined by qRT-PCR. Levels
of gli3 expression were normalized by actin expression. Note that no significant changes of gli3 mRNA
expression in ptch1-deficient GNPs and NIH3T3 cells with altered Nestin expression. D-E, Cell cycle
distribution in NIH3T3 cells with altered Nestin expression (D), in Nestin+ and Nestin- ptch1-deficient
GNPs (E), was analyzed by flow cytometry.
Supplementary Figure S4 Nestin knockdown in MB cells by Nestin shRNA
Expression of Nestin, RFP and Actin proteins in MB cells after infection with lentivirus carrying scrambled
shRNA or Nestin shRNAs (#4 or #5), examined by western blotting.
Supplementary Figure S5 Proliferation and Hh signaling in MB cells at 24hrs after Nestin depletion
MB cells were infected with lentivirus carrying scrambled shRNA or Nestin shRNA in vitro. Infected cells
(RFP+) purified by FACS at 24hrs after infection, were immunostained for Ki67 and RFP (A-B), and
analyzed for mRNA expression of gli1, cyclinD1 and n-myc by qRT-PCR (C).
Supplementary Figure S6 Ciliogenesis among GNPs and NIH3T3 cells after Nestin expression.
A, NIH3T3 cells with altered Nestin expression were starved for 16hrs to induce ciliar formation, and then
treated with medium containing 10% fetal bovine serum for 2hrs to disassemble cilia (2). Before and after
serum treatment, NIH3T3 cells were immunostained with an antibody against Arl13b (3), to label primary
cilia. The percentage of cells with cilia among Nestin-overexpressed (Nes O/E) and Nestin-deficient (Nes
shRNA) NIH3T3 cells was quantified. B, Cerebellar sections from P7 Math1-Cre/ptch1C/C/Nestin-CFP
mouse were immunostained for CFP and Arl13b, and counterstained with DAPI. C, The percentage of cells
with cilia among CFP+ cells and CFP- ptch1-deficient GNPs were quantified.
Supplementary Figure 7 Nestin expression in human MB
A, Nestin expression in human MBs was examined by immunohistochemistry using tissue microarrays. BC, Representative images of Nestin+ (b) and Nestin- (c) human MB. d, Summary of Nestin expression
among 287 human MBs analyzed by tissue microarrays.
Supplementary Table 1 Gene differentially expressed between Nestin+ vs Nestin- ptch1-deficient
GNPs (ratio ≥2.5-fold, p<0.01)
Gene name
Mu-crystallin homolog
PDZ and LIM domain 3
Thrombospondin, type I, domain containing 7B
Nestin
Glutamic acid decarboxylase 1
Lysozyme 1
Potassium voltage-gated channel, member 1
SKI family transcriptional co-repressor 1
Zinc finger matrin type 4
Chemokine (C-X-C motif) ligand 12
Ryanodine receptor 2, cardiac
Glutamate receptor, metabotropic 1
L1 cell adhesion molecule
Eph receptor B1
Neurotrophic tyrosine kinase receptor type 2
Phosphotyrosine interaction domain containing 1
Opioid receptor-like 1
Placenta-specific 8
Zinc finger and BTB domain containing 46
Multiple EGF-like-domains 11
Semaphorin-3D
Potassium voltage-gated channel subfamily D member 2
Cell adhesion molecule 2
Tetraspanin 4
Transcription factor AP-2 alpha
Arginase type II
Gene symbol
Fold change
Crym
Pdlim3
Thsd7b
Nes
Gad1
Lyz1
Kcna1
Skor1
Zmat4
Cxcl12
Ryr2
Grm1
L1cam
Ephb1
Ntrk2
Pid1
Oprl1
Plac8
Zbtb46
Megf11
Sema3d
Kcnd2
Cadm2
Tspan4
Tcfap2a
Arg2
6.7↑
5.2↑
4.3↑
2.8↑
7.7↓
7.7↓
4.6↓
4.0↓
3.6↓
3.6↓
3.2↓
3.0↓
3.0↓
3.0↓
2.8↓
2.8↓
2.8↓
2.7↓
2.7↓
2.7↓
2.6↓
2.6↓
2.6↓
2.6↓
2.6↓
2.5↓
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1.
Pugacheva EN, Jablonski SA, Hartman TR, Henske EP, Golemis EA. HEF1dependent Aurora A activation induces disassembly of the primary cilium. Cell.
2007;129:1351-63.
2.
Higginbotham H, Guo J, Yokota Y, Umberger NL, Su CY, Li J, et al. Arl13bregulated cilia activities are essential for polarized radial glial scaffold formation. Nat
Neurosci. 2013;16:1000-7.