Molecular CelL, Volume 44

Supplemental Information

NSD2 Links Dimethylation of Histone H3

at Lysine 36 to Oncogenic Programming

Alex J. Kuo, Peggie Cheung, Kaifu Chen, Barry M. Zee, Mitomu Kioi, Josh Lauring, Yuanxin Xi, Ben Ho

Park, Xiaobing Shi, Benjamin A. Garcia, Wei Li, and Or Gozani

Figure S1, Related to Figure 1. NSD2 Catalyzes Dimethylation of H3K36 In Vitro

(A) Schematic of NSD2 protein showing the locations of NSD2SET constructs, shRNA-targeting

sequences and inactivating mutations used in this study. Top: schematic of full length NSD2 protein.

Evolutionarily conserved protein motifs are highlighted. The two independent shRNAs targeting the

NSD2SET domain are shown in pink. The NSD2SET construct is depicted in blue. Bottom: sequence

alignment of NSD2SET with other PKMT SET domains. Blue background indicates the two conserved

tyrosine residues that are mutated to alanine. Y1092: predicted catalytic site; Y1179: predicted SAM and

target lysine binding site (Dillon et al., 2005).

(B) Full-length NSD2 methylates histone H3 on native nucleosomes. Autoradiogram of methylation assay

on HeLa nucleosomes with full-length NSD2 purified from HT1080 cells stably transduced with FLAG-

NSD2. Nucleosome only reaction in lane 1 lacks PKMT. Control reaction in lane 2 uses IPed material

from HT1080 cells transduced with empty vector.

(C) NSD2 mono- and di-methylates H3K36 on native nucleosomes. Western analysis of methylation

assay performed on HeLa nucleosomes ± full-length NSD2 purified from HT1080 cells as in (B), using

the indicated antibodies.

(D) Alternatively spliced NSD2 variant REIIBP di-methylates H3K36 on native nucleosomes. Western

analysis of methylation assay performed on HeLa nucleosomes with NSD2SET or isoform REIIBP.

Control reaction lacks PKMT.

(E) NSD2 shows no methylation activity on histone H4. Autoradiogram of methylation assay on

recombinant H4 tail library (aa 1-40) with NSD2SET and SETD8. Bottom panel shows a Coomassie stain

of the H4 tail substrates.

(F) PTM-specific antibodies used in this study recognize the target epitopes but not other modifications

on histones. Epigenome microarray analysis of the rabbit polyclonal antibodies against H3K36me2 (top

panel) and H3K36me3 (bottom panel). 83 histone peptides carrying distinct PTMs are printed in 6

replicates on the array. The red spots indicate the specific interactions between antibodies and the target

epitopes. (G) t(4;14) translocation causes increase in NSD2 expression. Western analysis of WCE from

KMS11 and two TKO cells using anti-NSD2 antibodies. H3 is used as a loading control. NSD2 signal

intensities are quantified and shown.

Figure S2, Related to Figure 3. t(4;14)-Driven NSD2 Overexpression Reprograms

H3K36me2 Genomic Distribution in Myeloma Cells

(A and B) t(4;14)-associated NSD2 overexpression abrogates intragenic enrichment of H3K36me2 in

t(4;14)+ myeloma cells. (A) Pie charts illustrate the distribution of H3K36me2-enriched peaks between

intergenic (yellow) and intragenic (purple) genomic regions in KMS11 and TKO2 cells. (B) Genomic

distribution of nucleotides associated with the indicated H3K36me2 ChIP-sequencing reads in KMS11

and TKO2 cells. Note that (A) and (B) are biological replicates of Figures 3B and 3C.

(C) t(4;14)-driven NSD2 overexpression disrupts TSS-proximal H3K36me2 enrichment in myeloma

cells. Average H3K36me2 profiles of 20,910 annotated genes in KMS11 and TKO2 cells. The scales of

the x- and y-axes are as in Figure 3D. The data represent a biological replicate of Figure 3D.

Figure S3, Related to Figure 4. t(4;14)-Induced NSD2 Overexpression Activates Oncogene

Expression in Myeloma Cells

(A) Depletion of NSD2 in KMS11 cells by RNAi correlates with global downregulation of transcription.

Genome-wide expression profiling of KMS11 cells stably expressing control shRNA in comparison to

KMS11 cells expressing two independent shRNAs targeting NSD2 (see Figure 2). Top: heatmap

representation of genes that are differentially expressed in NSD2 knockdown versus control cells (n=2 for

each NSD2 RNAi). Numerical expression values for each gene were median centered and scaled to range

from -1.0 (blue; low expression) to 1.0 (yellow; high expression). Bottom: overlap of differentially

expressed genes between the two NSD2 RNAi cell lines. p-values: statistical significance of the overlap.

Activated: downregulated in NSD2-RNAi/control cells; repressed: upregulated in NSD2-RNAi/control

cells.

(B) t(4;14)-induced NSD2 overexpression alters transcription profiles of myeloma cells. Quantitative RT-

PCR analysis of mRNA isolated from KMS11 cells and two independently generated TKO lines. “NSD2:

Activated” and “NSD2: Repressed” are as in Figure 4B.

(C) Positive correlation between H3K36me2 levels and transcription is disrupted in t(4;14)+ myeloma

cells. Average H3K36me2 profile of genes divided into three expression quantiles. The expression cutoffs

are as in Figure 4C. The data represent a biological replicate of Figure 4C.

(D) t(4;14)-induced H3K36me2 reprogramming is associated with differential gene expression profiles of

myeloma cells. Average H3K36me2 distribution of group A and group B genes, as defined in Figure 4E.

The data represent a biological replicate of Figure 4D.

(E) The transcript levels of a subset of genes in multiple myeloma patients positively or negatively

correlate with NSD2 expression. Heatmaps of the genes whose transcription level positively or negatively

correlates with NSD2 expression using Pearson correlation coefficient cutoff= 0.3.

(F) Direct binding of NSD2 at oncogenes is linked to transcriptional activation in t(4;14)+ myeloma cells.

ChIP analysis of NSD2 occupancy at two oncogenes, MET and RRAS2, in KMS11 and TKO2 cells. Top:

schematics of the two genes. Numbers indicate the genomic locations of primer pairs. Middle two insets:

snapshots of H3K36me2 ChIP-sequencing signals using custom tracks in the UCSC genome browser.

Bottom insets: NSD2 ChIP signals assessed by quantitative real-time PCR using the indicated primer

pairs. Control ChIP utilizes uncoupled protein A beads.

Figure S4, Related to Figures 5, 6, and 7. NSD2 Promotes Cancer-Related Cellular

Processes in a Methyltransferase Activity-Dependent Manner

(A) Y1092A and Y1179A mutations abrogate NSD2 lysine methyltransferase activity. Top:

autoradiogram of methylation assay with wild-type NSD2SET or catalytically inactive mutants on native

nucleosomes. Y1118A mutation has been reported to disrupt NSD2 enzymatic activity (Martinez-Garcia

et al.). Middle and bottom: Coomassie stain of NSD2SET proteins (middle) and HeLa nucleosomes

(bottom).

(B) Depletion of NSD2 slows the proliferation rate of t(4;14)+ myeloma cells. Growth rate analysis of

KMS11 cells stably transduced with control vector or two independent shRNA targeting NSD2. Error

bars represent s.e.m. from three replicates.

(C) Overexpression of NSD2 and intact NSD2 catalytic activity are required for myeloma cell growth in

mouse xenograft model. Intravenous injection of modified myeloma cells into SCID-Beige mice as in

Figure 6D. Bioluminescent images show tumor growth of all mice in the experiment.

(D) NSD2 supports tumor growth of non-myeloma cancer cells. Growth curve analysis of human

fibrosarcoma cell line HT1080 (top) and osteosarcoma cell line U2OS (bottom) treated with control small

interfering RNA (siRNA) or two NSD2-targeting siRNAs.

(E) Elevation of global H3K36me2 levels in t(4;14)+ myeloma cells. Western analysis of WCE from

myeloma cell lines with or without t(4;14) chromosome translocation. Asterisk (*) indicates full-length

NSD2.

(F) Ectopic NSD2 induces oncogenic transformation of p19ARF-/- MEFs. Top: western analysis of WCE

from p19ARF-/- MEFs stably transduced with control vector, wild-type NSD2 or NSD2 Y1179A mutant.

Bottom: soft agar colony formation assay to test anchorage-independent growth of the indicated modified

p19ARF-/- MEFs. Error bars represent s.e.m. from three replicates. The data represent a biological replicate

of Figure 7F.

(G) Ectopic NSD2 alters global gene transcription profiles of p19ARF-/- MEFs. Genome-wide expression

profiling of p19ARF-/- MEFs stably transduced with control vector or NSD2WT. Heatmap shows differential

genes in NSD2WT-expressing cells relative to control cells identified using the dChip program (n=3;

>1.25 fold) (Li and Hung Wong, 2001).

Figure S5, Related to Figure 7 and Discussion. NSD2 Is a General Oncoprotein and Not

Involved in the Classical Tumor-Suppressive DNA Damage Response Pathways

(A) H3K36 and H4K44 are highly similar in sequence. Alignment of amino acid sequences flanking

H3K36 and H4K44. H3K36 and H4K44 are highlighted in blue.

(B) Model for how NSD2 H3K36 dimethylation activity contributes to oncogenic programming. Top

Schematic: Normal expression of NSD2 (t(4;14)-negative cells) results in appropriate binding of NSD2 to

physiologic target genes (Class I) and subsequent dimethylation at H3K36 throughout the transcribed

body, with TSS-proximal nucleosomes being most heavily enriched with H3K36me2. This activity is

associated with expression of Class I genes, which in the case of plasma cells, regulate normal B cell

physiologic pathways. In the t(4;14)-negative cells, a number of genes that can promote oncogenesis

(Class II, e.g. c-Met) are not active. Bottom Schematic: Pathologic NSD2 overexpression due to the

t(4;14) translocation overwhelms the mechanisms involved in proper targeting of NSD2 and results in

aberrant and deregulated dimethylation at H3K36 throughout the genome. The efficiency of Class I gene

transcription is diminished and Class II genes are inappropriately activated. The combined action of

lower Class I transcription coupled to increased expression of Class II genes selects for a gene expression

program that is favorable for myelomagenesis. This mechanism could also contribute to onogenic

programming in diverse cancer types. Red flags represent H3K36me2.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Reagents, Plasmids, and Antibodies

The cloning of full-length NSD2, NSD2SET and isoform REIIBP was based on NCBI

sequence NM_001042424.2. NSD2SET was amplified by the primer pairs 5’-

CGCGGATCCAAAAACGCATTGCAAG-3’/5’-CGCCTCGAGTTACTATTTGCCCTCTG-3’.

REIIBP was amplified using the primer pairs: 5’-

CTGGGATCCATGATGCGGTGTGTCCGCTGC-3’/5’-

CGCCTCGAGTTACTATTTGCCCTCTG-3’. NSD2SET and REIIBP PCR fragments were

cloned into pGEX-6-P1 using BamHI/XhoI sites. H4 tail library was generated by cloning the

oligos corresponding to H4 amino acids 1-40 with all the lysines replaced by arginines into

pGEX-6-P1(GE Healthcare). Each arginine was restored back to lysine by site-direct

mutagenesis. Full-length NSD2 cDNA was cloned into pCAG-FLAG for overexpression in 293T

cells; pMSCV vectors with 3X-FLAG-MYC epitopes for retroviral transduction of HT1080

cells; and pENTR3C/pDEST20 (Invitrogen) for the purification of recombinant full-length

NSD2 from Sf9 cells. For lentiviral transduction, full-length NSD2 was cloned into

pENTR3C/pLenti6.2/V5-DEST (Invitrogen), and viral particles were prepared with packaging

plasmids pCMV∆8.91 and pMD.G. NSD2 mutants were generated by site-directed mutagenesis

(Stratagene). The luciferase-GFP cassette (a gift from Dr. C.G. Fathman (Stanford University))

was subcloned into pENTR3C and recombined into pLenti6.2/V5-DEST/puromycin (Cordero

Lab, Stanford University) for lentiviral transduction. Antibodies used in the study were: H3,

H3K4me2, H3K9me2, H3K36me1, H3K36me3, H4K20me1, H4K20me2, H4K20me3 (Abcam);

H3K36me3 (Cell Signaling); H3K36me2, H3K27me2, H3K4me3 (Active Motif); H3K27me3

(Millipore); FLAG(M2) (Sigma); GFP (Enzo Life Sciences).

In Vitro Methylation Assays

Methylation assays were performed as described (Shi et al., 2007) using 1 g

recombinant or purified nucleosomes or 1 g of recombinant histone.

Cell Culture and Transfection

KMS11, TKO and modified TKO cells were cultured in RPMI1640 supplemented with

10% fetal bovine serum (ATCC), glutamine (Gibco) and penicillin/streptomycin (Gibco). 293T,

HT1080, U2OS and p19ARF-/-

MEFs were cultured in DMEM with 10% fetal bovine serum and

supplements. H929, KMS12PE, U266 and RPMI8226 were kept in RPMI1640 containing 10%

newborn calf serum (Gibco) and supplements. Transfection of plasmid DNA was performed

using TransIT-LT1 or TransIT-293 (Mirus) following the manufacturer’s protocol. HT1080 and

U2OS were transfected with siRNA targeting NSD2 transcripts using Dharma-FECT

(Dharmacon) transfection reagent. p19ARF-/-

MEFs were a gift from S. E. Artandi (Stanford

University). For growth curve analysis, cells were plated in triplicate and counted every 2 days

after seeding.

Baculovirus Expression of Full-Length NSD2

Generation and purification of recombinant full-length NSD2 were performed following

the manufacturers’ manuals (Invitrogen). In short, pDEST20-NSD2 was utilized to transform

DH10Bac E. coli to obtain shuttle bacmid. Sf9 cells were transfected with bacmid DNA to

produce baculovirus particles and the virus titer was amplified through multiple rounds of

transduction. Log-phase Sf9 cell culture (106/mL) was transduced with virus stock and cells were

harvested 2 days after transduction. The GST-tagged recombinant proteins were purified using

glutathione-conjugated agarose (Amersham), and the quality of the purified proteins was

monitored by SDS-PAGE followed by Coomassie staining.

Purification of Recombinant Full-Length NSD2 in Mammalian Cells

HT1080 cells transduced with pMSCV-FLAG-MYC-NSD2 or control vector were

generated. Whole cell extract was prepared using buffer A containing 50mM Tris-HCl (pH7.4),

250mM NaCl, 0.5% Triton X-100, 10% glycerol, 1mM PMSF. Full-length NSD2 was affinity-

purified with anti-FLAG(M2)-conjugated agarose (Sigma) and eluted with 0.4mg/ml FLAG

peptides for in vitro methylation assays.

RNAi Knockdown Strategy

Oligos targeting NSD2 or SETD2 SET domain were synthesized and cloned into

pLentiLox3.7 to generate lentivirus particles for stable transduction. The two shRNAs target

NSD2 transcripts at 5’-GGACATCAGAAAGGGAGAATT-3’ (shRNA#1) or 5’-

GGAAACTACTCTCGATTTATG-3’ (shRNA#2). SETD2 shRNA targets 5’-

GAGAGGTACTCGATCATAAAGAGTT-3’ at SETD2 transcript. Transduced KMS11 cells

were selected with 10ug/mL puromycin. The two siRNAs targeting the SET domain of NSD2

transcripts, siRNA#1: 5’-GCACACGAGAACGACATCA-3’ and siRNA#2: 5’-

CCACTTCTACATGCTCA-3’, were applied to deplete NSD2 in HT1080 and U2OS cells.

Quantitative Mass Spectrometry

Acid-extracted total histones were subjected to chemical derivatization using D0-proionic

anhydride followed by digestion with trypsin at a substrate:enzyme ratio of 10:1 for 6 hours at

37°C as previously described (Plazas-Mayorca et al., 2009). For comparative analysis of both

KMS11 and TKO2, we then performed a second round of propionylation on the digested

peptides, with one sample being derivatized with the same D0-propionic anhydride reagent and

the other sample being derivatized with D10-propionic anhydride for quantitative proteomics as

previously described (Plazas-Mayorca et al., 2009). Using D10-propionic anhydride introduces a

5 Da shift by derivatization of the free N-termini of all peptides generated from the trypsin

digest. Equal amounts of both samples as quantified by a Bradford assay were mixed together,

and digested peptides were desalted using homemade STAGE tips as reported earlier (Rappsilber

et al., 2003) Desalted peptides were loaded onto fused silica microcapillary column (75mm)

packed with C18 rein constructed with an ESI tip through an Eksigent AS-2 autosampler

(Eksigent Technologies Inc.) at a rate of 200 nL/min. Peptides were eluted using a 5-35% solvent

B in 60 minute gradient (solvent A= 0.1 M acetic acid, solvent B = 70% acetonitrile in 0.1 M

acetic acid) Nanoflow LC-MS/MS experiments were performed on an Orbitrap mass

spectrometer (ThermoFisher Scientific) taking a full mass spectrum at 30,000 resolution in the

Orbitrap and 7 data-dependent MS/MS spectra in the ion trap. All MS and MS/MS spectra were

manually verified.

Chromatin Immunoprecipitation (ChIP) Analysis

ChIP was performed using the corresponding antibodies and protein A-conjugated

magnetic beads (Invitrogen). Briefly, cells were cross-linked with 1% formaldehyde and lysed

with the buffer containing 50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS and protease

inhibitors. Cell lysate was sonicated with Branson Digital Sonifier (Branson) and diluted with

RIPA buffer by 10 fold (v/v) before incubating overnight with antibody/protein A-magnetic bead

complexes. In the next day, beads were washed three times with RIPA buffer and twice with TE

buffer. IPed DNA was eluted from beads, de-crosslinked at 68 C and purified using the

MinElute PCR Purification Kit (Qiagen). Purified DNA was subject to ChIP-sequencing analysis

or quantitative real-time PCR analysis. For RT-PCR analyses, purified ChIP DNA was analyzed

on LightCycler 480 (Roche) using SYBR Green Master Mix (Roche) following the

manufacturer’s manual. All samples were normalized to input as control. Sequences of primer

pairs used in the study are listed below.

TGFA Promoter:

5’-GGAGCTCACGGACTGACC-3’

5’-GATCTACCCGGAGCACGTAG-3’

TGFA TSS:

5’-CGTAGCCGGATTGTCCTG-3’

5’-GCCCCTCGGTGTAGGTAAC-3’

TGFA (1) 6kb:

5’-CAAGATGAAAACAGGTGAAGC A-3’

5’-TCAAGACGGAACCCCTTTAG-3’

TGFA (2) 22kb:

5’-GTTGAAAGCGACGAAACCAT-3’

5’-GTGCTGGTTTGGGTTTTCTC-3’

TGFA (3) 57kb:

5’-CTGGAAAAACTGGGGAAAGA-3’

5’-TTCTAGGCTTTGCACTTGCTC-3’

TGFA (4) 73kb:

5’-TCCTGCCTCTTCCATGATCT-3’

5’-AAACCAGGAATTCCCAACTTTT-3’

TGFA (5) 101kb:

5’-AGAGGTGAGTAACTTGTCAAAGGTC-3’

5’-GTAGGGACCCCTTACATCCTG-3’

PAK1 Promoter:

5’-TGTTTTCTTTCTGTGGGAGAGG-3’

5’-CACATTAGTTCAAACATCTCCGTTA-3’

PAK1 TSS:

5’-GAGGGGGCGTCTACTGTG-3’

5’-GTACAATAGCGCGGCTGTG-3’

PAK1 (1) 7kb:

5’-CACCCCCATTTCAAGTGC-3’

5’-TAAGTGCAAGTGCCATGTGAA-3’

PAK1 (2) 47kb:

5’-CAGAACAAGAAAAGTTCAAGATGC-3’

5’-ATCCCACTACCAACCCCATT-3’

PAK1 (3) 90kb:

5’-CCAGATTCACAAAGCACATGA-3’

5’-TCCCTCAGTCCGAAGCTCT-3’

PAK1 (4) 124kb:

5’-TTTCCTGTTCCTGCTTGCTT-3’

5’-AATTAGAGCCACGTGCCAAG-3’

MET Promoter:

5'-GGAGACTCGGTCCCGCTTAT-3'

5'-CCCAGCTCAGGCAGTCTGA-3'

MET TSS:

5’-TGACACTCGCCTCCCAAG-3’

5’-AAGTTAGCACAGCCGGAGATA-3’

MET (1) 3kb:

5’-CGTTTCTTCTTTAGGCATTAGGC-3’

5’-ACCACGGAAAAGAAAGCGTA-3’

MET (2) 26kb:

5’-TGGTGCAGAGGAGCAATGG-3’

5’-CCCAGTCTTGTACTCAGCAAC-3’

MET (3) 45kb:

5’-GTCATCACCACGAGGCTGT-3’

5’-GTGATTCACAAGGTGATGGAAG-3’

MET (4) 71kb:

5’-GCTCGCAGCAAGATCAGTG-3’

5’-CCGTGTACCTCTGTTGGACA-3’

MET (5) 114kb:

5’-GGAGAAATTGGATGCTCAACA-3’

5’-TTCCAAGACCCTTCTGGTGT-3’

RRAS2 Promoter:

5’-AGTGGGTGTCAGTTGGGAGT-3’

5’-CCACACAATCCCTTACATAGACAA-3’

RRAS2 TSS:

5’-CCAAGTTGCCACCGCTAT-3’

5’-AGCCGGGCTTTACTGCTC-3’

RRAS2 (1) 5kb:

5’-CAAATCTCCTGAAATCTCTTCTCG-3’

5’-TGCTGAGTACTTTTTCATTGCTTT-3’

RRAS2 (2) 15kb:

5’-CACGCCTGTACTCCCAGTTAC-3’

5’-TGTCCCCGGCTTAAGTGAT-3’

RRAS2 (3) 24kb:

5’-GGTCCTTGCCATCAACAACT-3’

5’-TGAGGAAAAAGCTAGTACAATAGGG-3’

RRAS2 (4) 50kb:

5’-AATGTGCCAAGCATTGTGTC-3’

5’-GAAACTGCCAGCCCTCTAAG-3’

RRAS2 (5) 74kb:

5’-CCATACCAATTCAAGTATGGTTTAAGA-3’

5’-GGGACTTTAGGCATACACCACT-3’

Reference Gene Sets

We used the human reference genome hg18 and RefSeq annotation with in total 34,237

genes. Among the 48,802 probe sets on the gene expression array (HumanHT-

12_V3_0_R2_11283641_A), 27,456 probe sets could be mapped to 20,910 high confidence

protein-coding (Accessions starting from “NM”) RefSeq genes, which were selected for

downstream analysis. The remaining 13,327 RefSeq genes are either low confidence genes or

Unigenes that lack exact gene structure.

H3K36me2 ChIP-Seq Data Analysis

In total, 130,438,736 raw reads were generated using Illumina Genome Analyzer II single

end sequencing protocol from six samples, including input materials from KMS11 and TKO2

cells and two biological replicates of H3K36me2 ChIP DNA. 48.09% of the raw reads passed

quality filtering and were uniquely mapped to the human genome using Illumina Eland software,

allowing up to 2 mismatches. Average size of ChIP-DNA fragment in each sample was

estimated by cross-strand Pearson correlation. The 5’ end of each uniquely mapped and high-

quality read was shifted half fragment size toward 3’ end, and then extended 50bp to both

directions. H3K36me2 signal intensity at each nucleotide was calculated as read coverage,

followed by scaling normalization to ensure that the average intensity across the whole genome

equals to 1 for each sample. The input intensity was subtracted from the ChIP signal based on a

Poisson model:

where st is the ChIP intensity and Si is the input intensity, St-c is the resulting ChIP intensity after

input subtraction, b is the background value provided as a parameter for normalization, and is set

to 1 in this work. Average profiles of H3K36me2 across gene body were all derived from these

input-subtracted and normalized intensity profiles.

Analysis of Multiple Myeloma Patient Datasets

Expression datasets of Multiple Myeloma Research Consortium (MMRC) patient

samples were downloaded from http://www.broadinstitute.org/mmgp/home. Genes whose

transcript levels correlated with NSD2 expression were identified using Pearson correlation

coefficient >0.3 (positively correlated) or <-0.3 (negatively correlated). p-values calculated by

Fisher’s exact test were used to measure the statistical significance of the overlaps between gene

lists. The heatmap shown in Figure S34 was plotted by Mev_4_4 (v10.2). Hierarchical clustering

was performed based on Pearson correlation and average linkage.

Real-Time PCR Gene Expression Analysis

RNA samples were extracted from cells using RNeasy Plus kit (Qiagen) and reverse

transcribed into cDNA using the SuperScript III First Strand Synthesis System (Invitrogen).

Quantitative real-time PCR analysis was performed on a Roche LightCycler 480 using the

manufacturer’s Universal Probe Library system. The primer sequences and the corresponding

fluorecein-labeled probes (Roche) are listed below.

TGFA:

5’-CCTGGCTGTCCTTATCATCAC-3’

5’-GGCACCACTCACAGTGTTTTC-3’

probe#74

RRAS2:

5’-GAGCAGCCCGGCTAGATATT-3’

5’-TGTTCTCTCATGGCTCCAAA-3’

probe#18

PAK1:

5’-GGTTTCAAGTGTTTAGTAACTTTTCCA-3’

5’-TTAGCTGCAGCAATCAGTGG-3’

probe#19

CHD2:

5’-GGAGATCCGATGAATTTAGGC-3’

5’-GCGGTGATCAGACATTCGT-3’

probe#18

CIRBP:

5’-TACAGAGACAGTTATGACAGTTACGC-3’

5’-GCCATTGGAAGGACGATCT-3’

probe#4

NOTCH1:

5’-CGGGGCTAACAAAGATATGC-3’

5’-CACCTTGGCGGTCTCGTA-3’

probe#52

IRF1:

5’-GGGCTGTCAGTTGATTCTGG-3’

5’-CTATGGCACATGCCTCAAAA-3’

probe#57

RERG:

5’-AACTTGCAGAGGACCGTAGC-3’

5’-TTGGAAGAGTCCACAATCCTG-3’

probe#80

GAPDH:

5’-GAGTCCACTGGCGTCTTCAC-3’

5’-TTCACACCCATGACGAACAT-3’

probe#45

GAPDH:

5’-AGCCACATCGCTCAGACAC-3’

5’-GCCCAATACGACCAAATCC-3’

probe#60

Fos (Mus musculus):

5’-GGCTCTCCTGTCAACACACA-3’

5’-GACCAGAGTGGGCTGCAC-3’

probe#26

Igf2 (Mus musculus):

5’-CGCTTCAGTTTGTCTGTTCG-3’

5’-GCAGCACTCTTCCACGATG-3’

probe#40

Figf (Mus musculus):

5’-AAGGGCCCAGGGACTCTAC-3’

5’-GGGGGACTTGAAAGGAAGTT-3’

probe#2

Mdk (Mus musculus):

5’-GAGTGTTCGGAGTGGACCTG-3’

5’-GCTCCAAATTCCTTCTTCCAG-3’

probe#3

Gapdh (Mus musculus):

5’-AGCTTGTCATCAACGGGAAG-3’

5’-TTTGATGTTAGTGGGGTCTCG-3’

probe#9

Gapdh (Mus musculus):

5’-GCCAAAAGGGTCATCATCTC-3’

5’-CACACCCATCACAAACATGG-3’

probe#29

The expression of MET was examined by the primer pairs 5’-TGGTGCAGAGGAGCAATGG-

3'; 5’-CCCAGTCTTGTACTCAGCAAC-3', using SYBR Green Master Mix (Roche). The

expression of BACE2 and SATB1 were analyzed by TaqMan Gene Expression Assays (Applied

Biosystems). Expression data of all genes were normalized to GAPDH (or Gapdh) levels.

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