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 DDCT 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 Line-1 pyrosequencing primers were obtained from Biotage; additional pyrosequencing primer sequences are included in Table 1.
Table 1-Pyrosequencing Primers
Oligo Name / Sequence 5' to 3' / Modification / Primer start location(Ensembl genome database) / Ensembl ID of genes / Numbers of CG sites analyzed
DAPK-pyro-f / GAGGGTAGTTTAGTAATGTGTTATAGG / GRCh37:9:90112483 / ENSG00000196730 / 9
DAPK-pyro-r / CCTCCCAACTACCCTACCAAA / 5'-biotin / GRCh37:9:90112689
DAPK-pyro-s / TTTAGTAATGTGTTATAGGT
CDH1-pyro-f / ATTTTAGTAATTTTAGGTTAGAGGGTTA / GRCh37:16:8771006 / ENSG00000039068 / 7
CDH1-pyro-r / ACCACAACCAATCAACAAC / 5'-biotin / GRCh37:16:8771112
CDH1-pyro-s / ATTTTAGGTTAGAGGGTTAT
DKK1-pyro-f / GGGTGAAGAGTGTTAAAGGTT / GRCh37:10:54073880 / ENSG00000107984 / 6
DKK1-pyro-r / AACCATCATCTCAAAAAAACTCA / 5'-biotin / GRCh37:10:54074205
DKK1-pyro-s / TTGTGTTTTTTGTAGTTA
RASSF1-pyro-f / AGTTTGGATTTTGGGGGAGG / GRCh37:3:50378342 / ENSG00000068028
(negative strain) / 5
RASSF1-pyro-r / CAACTCAATAAACTCAAACTCCCC / 5'-biotin / GRCh37:3:50378206
RASSF1-pyro-s / GGGTTAGTTTTGTGGTTT
NBL2-pyro-f / AGTAGTTGGTGTTAATGTGTGTTA / NA / 4
NBL2-pyro-r / ACTCTCTATATATTTCTTTCCCACT / 5'-biotin
NBL2-pyro-s / ATTAGAGGAGTAAAGAGGTT
D4Z4-pyro-f / GAGTTCGGAGTTTTTGTAGTAGG / NA / 4
D4Z4-pyro-r / AAAAATCCCAAACCCATCAACC / 5'-biotin
D4Z4-pyro-s / GTAGGAGTAATTTTCTTTAGA
HSPB1-pyro-f / GTGTGGTTTTAATTTTGTTTTTGTTATTT / GRCh37:7:75931609 / ENSG00000106211 / 8
HSPB1-pyro-r / ATTCAACCCTCATCTAAAACCTTCTCT / 5'-biotin / GRCh37:7:75931832
HSPB1-pyro-s / AGGGTTATAGTTAGTAAAGTTTAAG
RAR-beta-pyro-f / TGTTAAAGGGGGGATTAGAAT / GRCh37:3:25469808 / ENSG00000077092 / 10
RAR-beta -pyro-r / AATAAATACTTACAAAAAACCTTCC / 5'-biotin / GRCh37:3:25469952
RAR-beta -pyro-s / TGTTTGAGGATTGGGAT
PLD5-pyro-f / TAGTAAAGATGAGTTAGGGAGAGGT / GRCh37:1:242688212 / ENSG00000180287
(negative strain) / 12
PLD5-pyro-r / AACCC(A/G)C(A/G)CCTACAAATTAATA / 5'-biotin / GRCh37:1:242688098
PLD5-pyro-s / GTTAGGGAGAGGTGTA
CDH13-pyro-f / TTTGGGAAGTTGGTTGGTTG / NT_010498.15 / 6
CDH13-pyro-r / ACAACCCCTCTTCCCTACCT / 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 / dbSNP accession No: rs680
IGF2-SNP-r / TCGGATGGCCAGTTTACCCTGAAA / 5'-biotin / NA
IGF2-SNP-seq / ACCAGCAAAGAGAAAAGAA
H19-SNP-f / TTTGCACTGGTTGGAGTTGTGGAG / dbSNP accession No: rs3741219
H19-SNP-r / GGCGTAATGGAATGCTTGAAGGCT / 5'-biotin / NA
H19-SNP-seq / GCCACCTTGGCAAGTGCCTG
* Sequence is confidential information of Qiagen
Chromatin immunoprecipitation (ChIP): Chromatin immunoprecipitation was performed using reagents and protocols contained in the Millipore 17-295 ChIP kit (Billeria, MA). Briefly, DNA-protein 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 / TGTCTGGCTCTGTCCTCCTTHSPB1-Chip-R / GTTCAGCCCTCATCTGGAAC
MyoD-Chip-F / CCTCTTTCGGTCCCTCTTTC
MyoD-Chip-R / TTCCAAACCTCTCCAACACC
GAPDH-Chip-F / TACTAGCGGTTTTACGGGCG
GAPDH-Chip-R / 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.
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 re-suspended 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 colony-formation 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 K-SFM 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.
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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