Supplementary Materials
RNA extraction and quantitative real-time RT-PCR. Total RNA was prepared using Trizol
reagents (Invitrogen, Carlsbad, CA) and RNeasy midiprep columns (Qiagen, Valencia, CA)
according to vendor instructions. RNA samples were quantified by Nano drop (Vendor,
Location), and their integrity and purity confirmed using an Agilent Bioanalyzer (Agilent, Foster
City, CA). 1 ug of total RNA was used for reverse transcription by using iScript cDNA Synthesis
Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. The resulting cDNA was
used in subsequent real time quantitative PCR and superarray PCR reactions. All RT-PCR
reactions were performed using ABI Taqman FAST master mix in an ABI 7900 Fast machine.
Data were analyzed using the CT method, normalized to ß-actin. Primer and probe sequences
used for qRT-PCR experiments are included in Table 1.
Genomic DNA isolation, bisulfite conversion, and pyrosequencing. Genomic DNA was isolated
from cells using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the
manufacturer’s protocol. 0.5 µg of DNA was used for bisulfite conversion by using EpiTect
Bisulfite Kit (Qiagen, Valencia, CA). Bisulfite-modified DNA was dissolved in 30 µl H2O and 2
µl of DNA template was used for pyrosequencing PCR amplification. Dispensation order
pyrosequencing reactions and data analysis were performed using the Pyromark MD
pyrosequencer with software provided by Biotage (Charlottesville, VA). p16, MGMT and Line1 pyrosequencing primers were obtained from Biotage; additional pyrosequencing primer
sequences are included in Table 1.
1
Table 1-Pyrosequencing Primers
Oligo Name
Sequence 5' to 3'
DAPK-pyro-f
GAGGGTAGTTTAGTAATGTGTTATAGG
DAPK-pyro-r
CCTCCCAACTACCCTACCAAA
DAPK-pyro-s
TTTAGTAATGTGTTATAGGT
CDH1-pyro-f
ATTTTAGTAATTTTAGGTTAGAGGGTTA
CDH1-pyro-r
ACCACAACCAATCAACAAC
CDH1-pyro-s
ATTTTAGGTTAGAGGGTTAT
DKK1-pyro-f
GGGTGAAGAGTGTTAAAGGTT
DKK1-pyro-r
AACCATCATCTCAAAAAAACTCA
DKK1-pyro-s
TTGTGTTTTTTGTAGTTA
RASSF1-pyro-f
AGTTTGGATTTTGGGGGAGG
RASSF1-pyro-r
CAACTCAATAAACTCAAACTCCCC
RASSF1-pyro-s
GGGTTAGTTTTGTGGTTT
NBL2-pyro-f
AGTAGTTGGTGTTAATGTGTGTTA
NBL2-pyro-r
ACTCTCTATATATTTCTTTCCCACT
NBL2-pyro-s
ATTAGAGGAGTAAAGAGGTT
D4Z4-pyro-f
D4Z4-pyro-r
GAGTTCGGAGTTTTTGTAGTAGG
AAAAATCCCAAACCCATCAACC
D4Z4-pyro-s
GTAGGAGTAATTTTCTTTAGA
HSPB1-pyro-f
GTGTGGTTTTAATTTTGTTTTTGTTATTT
HSPB1-pyro-r
ATTCAACCCTCATCTAAAACCTTCTCT
HSPB1-pyro-s
AGGGTTATAGTTAGTAAAGTTTAAG
RAR-beta-pyro-f
TGTTAAAGGGGGGATTAGAAT
RAR-beta -pyro-r
AATAAATACTTACAAAAAACCTTCC
RAR-beta -pyro-s
TGTTTGAGGATTGGGAT
PLD5-pyro-f
TAGTAAAGATGAGTTAGGGAGAGGT
PLD5-pyro-r
AACCC(A/G)C(A/G)CCTACAAATTAATA
PLD5-pyro-s
GTTAGGGAGAGGTGTA
CDH13-pyro-f
TTTGGGAAGTTGGTTGGTTG
CDH13-pyro-r
ACAACCCCTCTTCCCTACCT
Primer start location
(Ensembl genome database)
Modification
Ensembl ID of genes
Numbers of CG
sites analyzed
ENSG00000196730
9
ENSG00000039068
7
ENSG00000107984
6
ENSG00000068028
(negative strain)
5
GRCh37:9:90112483
5'-biotin
GRCh37:9:90112689
GRCh37:16:8771006
5'-biotin
GRCh37:16:8771112
GRCh37:10:54073880
5'-biotin
GRCh37:10:54074205
GRCh37:3:50378342
5'-biotin
GRCh37:3:50378206
5'-biotin
NA
4
5'-biotin
NA
4
GRCh37:7:75931609
5'-biotin
GRCh37:7:75931832
ENSG00000106211
8
ENSG00000077092
10
ENSG00000180287
(negative strain)
12
NT_010498.15
6
GRCh37:3:25469808
5'-biotin
GRCh37:3:25469952
GRCh37:1:242688212
5'-biotin
GRCh37:1:242688098
5'-biotin
CDH13-pyro-s
AGGAAAATATGTTTAGTGTA
P16-pyro-primer*
Qiagen cat no # 972012
NA
7
Line-1-pyro-primer*
Qiagen cat no # 973043
NA
4
MGMT-pyro-primer*
Qiagen cat no # 972032
NA
6
IGF2-SNP-f
CGAATTGGCTGAGAAACAATTGGC
IGF2-SNP-r
TCGGATGGCCAGTTTACCCTGAAA
IGF2-SNP-seq
ACCAGCAAAGAGAAAAGAA
H19-SNP-f
TTTGCACTGGTTGGAGTTGTGGAG
H19-SNP-r
GGCGTAATGGAATGCTTGAAGGCT
H19-SNP-seq
GCCACCTTGGCAAGTGCCTG
5'-biotin
dbSNP accession No:
rs680
NA
5'-biotin
dbSNP accession No:
rs3741219
NA
* Sequence is confidential information of Qiagen
2
Chromatin immunoprecipitation (ChIP): Chromatin immunoprecipitation was performed using
reagents and protocols contained in the Millipore 17-295 ChIP kit (Billeria, MA). Briefly, DNAprotein complexes were cross-linked with formaldehyde at a final concentration of 1% for 15
minutes. Immune complexes were formed with either non-specific IgG, or ChIP-grade antibodies
recognizing H3K9Ac (ab4441-50; Abcam, Cambridge, MA), or H3K27me3 (07-449; Millipore,
Billerica, MA). DNA was eluted and purified from complexes, followed by PCR amplification of
a region of the HSPB1 promoter adjacent to the transcription start site; primer sequences for
ChIP PCR are listed in Table 2.
Table 2- ChIP PCR Primers
HSPB1-Chip-F
HSPB1-Chip-R
TGTCTGGCTCTGTCCTCCTT
GTTCAGCCCTCATCTGGAAC
MyoD-Chip-F
MyoD-Chip-R
CCTCTTTCGGTCCCTCTTTC
TTCCAAACCTCTCCAACACC
GAPDH-Chip-F
GAPDH-Chip-R
TACTAGCGGTTTTACGGGCG
TCGAACAGGAGGAGCAGAGAGCGA
RT-PCR superarrays. Wnt signaling RT-PCR superarrays (PAH-043A) and Human Stress and
Toxicity Pathway Finder (PAH-003A) were obtained from SABiosciences (Frederick, Md). 1 ug
of total RNA was used for reverse transcription and the entire cDNA reaction was diluted and
distributed amongst the 96 wells of the superarray plate. The reactions were performed with RT²
SYBR Green / ROX PCR Master Mix (SABiosciences). The results were analyzed using
software provided by the vendor http://www.sabiosciences.com/pcr/arrayanalysis.php.
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Western blot analysis of histones: Western blot analysis of histone proteins was performed
according to protocols from Millipoe, with minor modifications. Briefly, cells were scraped from
10cm plates and centrifuged at 200Xg for 5 minutes. Cell pellets were washed with PBS and resuspended with 200ul Null buffer (10 mM HEPES, PH 7.9, 1.5 mM MgCL2, 10 mM KCL),
supplemented with 1X protease inhibitor cocktail (Roche, Indianapolis, IN). Thereafter, 20ul of
2N hydrochloric acid was added to the cell lysates on ice for 30 minutes, following which the
lysates were centrifuged at 11,000 x g for 10 minutes at 4 ºC. 20ug of acid-soluble supernatant
were used for western blot. The following antibodies were used: H4K16Ac (Abcam ab1762
Cambridge, MA), H4K20Me3 (Upstate 07-463 Billerica, MA), H3K27Me3 (Upstate 07-449),
total H3 (Upstate 05-499) and total H4 (Abcam ab10158). Western blot signals were detected
with the appropriate secondary-HRP conjugated antibodies and ECL reagent (Amersham,
Pittsburgh, PA), followed by densitometric analysis.
Soft-agar assays. Anchorage-independent growth of HBEC cells was determined by colonyformation efficiency in soft-agar. Briefly, 5 x103 control or 1.0% CSC-treated HBEC were mixed
at 37°C with 3ml/well of 0.4% (w/v) of soft-agar (Cell Biolabs, San Diego, CA) in K-SFM
medium and then poured onto a layer of previously plated 2ml/well of 0.6% soft-agar (w/v) in KSFM medium in six well plates. Cells were incubated for 3 weeks without CSC, with media
changed every 3 days. Soft agar colonies were counted using an inversion microscope at 4X
magnification. Randomly-selected soft agar colonies were isolated, expanded in the presence or
absence of CSC, and frozen for further studies.
4
Murine xenograft experiments. Control HBEC or CSC-derived soft agar clones were harvested,
washed, and suspended at 2 x 106 cells/ 200ul PBS, and inoculated subcutaneously into flanks of
athymic nude, or NOD.SCID\IL-2Rγ mice. After 12 weeks, mice were euthanized, and
evaluated for percent tumor take and tumor mass. Two independent experiments having a total of
100 animals were performed using each murine host. All animal procedures were approved by
NCI Animal Care and Use Committee, and were in accordance with the NIH Guide for the Care
and Use of Laboratory Animals.
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