Supporting InformationMertz et al. 10.1073/pnas.1108190108SI MethodsCell Cycle Analysis and Viability Determination. Cells were plated at5,000 cells per well of 96-well plates containing titrations of thecompounds as indicated. After incubation, the cells were washedonce with PBS and resuspended in 175 μL of ice cold 70%ethanol and fixed for a minimum of 16 h at 4 °C. The cells werepelleted and washed 1× with PBS and stained for 30 min at roomtemperature (RT) with 120 μL of staining solution [propidiumiodide (20 μg/mL; Sigma), RNase A (25 μg/mL; Sigma), 0.1%Triton X-100 in PBS].Cell number and cell cycle data were obtained by using a Guava

Easycyte flow cytometer using the Express Pro module. DNAcontent histograms were analyzed by using ModFit LT 3.2Software (Verity). To calculate the number of viable cells in eachwell, the concentration of events measured using the Guava wasmultiplied by the volume of cells in the well, then by the fraction ofcells in G1+S+G2/M. GI50 values for each cell line were calcu-lated as the concentration of compound giving a 50% reductionin cell number relative to the DMSO control.

Annexin V/PI Staining. After compound treatment, cells in 96-wellplates were washed once with PBS and stained with 50 μL ofAnnexin V-FITC/PI staining solution (Trevagen/R&D Sciences)in 1× binding buffer. After a 15-min incubation at RT, 70 μL ofbinding buffer was added to each well, and the cells were ana-lyzed by using a Guava Easycyte flow cytometer and the ExpressPro module. The percentage of viable cells (low annexin V-FITC/low PI), early apoptotic cells (high annexin V-FITC/lowPI), and late apoptotic/necrotic cells (high annexin V-FITC/highPI) was determined.

Gene Expression Profiling. LP-1 and Raji cells were treated with(+)-JQ1 or (-)-JQ1 at 500 nM for 4 or 8 h. Total RNA wasprepared by using a Qiagen RNEasy kit with on-column DNasedigestion according to the manufacturer’s instructions. Furtherprocessing of samples and data acquisition were performed atALMAC Diagnostics. Total RNA was amplified by using theApplause WT Amp Plus ST Amplification System (NuGEN)according to the manufacturer’s instructions. A modification steptargeting the amplified single-stranded cDNA resulted in thegeneration of sense target (ST) cDNA product compatible withthe Affymetrix GeneChip Exon arrays. The ST-cDNA was pu-rified for accurate quantitation and to ensure optimal perfor-mance during the fragmentation and labeling process. Theappropriate amount of amplified ST-cDNA was fragmented andlabeled by using the NuGEN Encore Biotin Module. The frag-mented and labeled ST-cDNA was added to the hybridizationmixture in accordance with the NuGEN guidelines for hybrid-ization onto GeneChip Exon 1.0 ST arrays (Affymetrix). Hy-bridization, washing, and array scanning were performedaccording to the manufacturer’s instructions. CEL files wereprocessed by the Affymetrix Expression Console softwarepackage by using the gene-level RMA algorithm on the core setof probesets and the annotation file Huex-1_0-st-v2_na31_hg19provided by Affymetrix. Detailed data analysis methods areavailable below.

Rescue Experiments. TET activator (pRETROX tet on advanced)and expression constructs (pRETROX tight puro luciferase/myc)were introduced into cells by viral transduction or FuGENE-mediated transfection. After selection, doxycycline was added for3 d and the cells were plated on 96-well plates containing a ti-

tration of compound as indicated. Viability and cell cycle dis-tribution were assessed as described above.

Cell Lysates and Western Blotting. Cells were harvested to obtainwhole-cell extracts by the addition of one volume of 250 mMTris·HCl (pH 6.8), 20% glycerol, 2% SDS, 5% 2-mercaptoe-thanol, and 0.2% bromophenol blue to cells in one volume ofPBS followed by boiling for 5 min. Samples were resolved onSDS/PAGE gels and transferred to PVDF membranes. Blotswere probed with antibodies to c-MYC (Cell Signaling) andvinculin (Sigma). After incubation with secondary antibodies(Jackson Immunoresearch or ThermoFisher), blots were de-veloped with ECL reagent (Perkin-Elmer) or analyzed on anOdyssey infrared imaging system (Licor).

Chromatin Immunoprecipitation (ChIP) Assays. Samples for ChIPanalysis were prepared by cross-linking cells with 1% formalde-hyde for 5 min at RT. Cross-linking reactions were quenched byaddition of glycine to a final concentration of 0.125 M. Cells werewashed with cold PBS and resuspended in lysis buffer (10 mMTris·HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2, and 0.2% NonidetP-40). After 1 h of incubation with rotation at 4 °C to lyse cells,nuclei were pelleted and resuspended in glycerol buffer (10 mMTris·HCl at pH 7.4, 0.1 mM EDTA, 5 mM MgOAc, and 25%glycerol) at a density of 5 × 106 cells per mL Genomic DNA wasfragmented to a size of 200–600 bp by treatment with micro-coccal nuclease (Roche). Nuclease digestions were stopped byaddition of EDTA to a final concentration of 10 mM. Lysateswere centrifuged to pellet debris and were precleared with pro-tein A or G beads that had been blocked with BSA and shearedsalmon sperm DNA. Precleared lysates were then used for im-munoprecipitations with the following antibodies: H4 tetra-ace-tyl (Active Motif), H3K4me3 (Abcam), H3 (Abcam), BRD3(Bethyl), BRD4 (Bethyl), CDK9 (Santa Cruz Biotechnology),and RSF (Millipore). After overnight incubation at 4 °C withrotation, protein A or G beads were added and incubated 1 h at4 °C with rotation. Beads were pelleted and washed once withlow salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris·HCl at pH 8, and 150 mM NaCl), once with high saltbuffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mMTris·HCl at pH 8, and 500 mM NaCl), once with LiCl buffer(0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mMEDTA, and 10 mM Tris-HCl at pH 8) and twice with T.E. (10mM Tris at pH 8 and 1 mM EDTA). Samples were eluted frombeads by two 15-min incubations at RT with elution buffer (1%SDS and 100 mM NaHCO3). Elutions were pooled, and NaClwere added to a final concentration of 0.2 M. After 4 h at 65 °C,DNA was purified from samples by using QIAquick PCR Puri-fication Kit (Qiagen). Purified DNA was used in PCRs withprimers in the MYC promoter region.ChIP analysis was also performed by Active Motif (Fig. S4) to

qualify the BRD4 antibody. ChIPs were performed as describedbelow for these experiments. Cells were fixed with 1% formal-dehyde for 15 min and quenched with 0.125 M glycine. Chro-matin was isolated by adding lysis buffer, followed by disruptionwith a Dounce homogenizer. Lysates were sonicated, and theDNA was sheared to an average length of 300–500 bp. GenomicDNA (Input) was prepared by treating aliquots of chromatinwith RNase, proteinase K, and heat for decross-linking, followedby ethanol precipitation. Pellets were resuspended, and the re-sulting DNA was quantified on a NanoDrop spectrophotometer.

Mertz et al. www.pnas.org/cgi/content/short/1108190108 1 of 13

Extrapolation to the original chromatin volume allowed quan-titation of the total chromatin yield.An aliquot of chromatin (25 μg) was precleared with protein A

agarose beads (Invitrogen). Genomic DNA regions of interestwere isolated by using 4 μg of antibody against BRD4. Afterincubation at 4 °C overnight, protein A agarose beads were usedto isolate the immune complexes. Complexes were washed,eluted from the beads with SDS buffer, and subjected to RNaseand proteinase K treatment. Cross-links were reversed by in-cubation overnight at 65 °C, and ChIP DNA was purified byphenol-chloroform extraction and ethanol precipitation.Quantitative PCRs (qPCRs) were carried out in triplicate on

specific genomic regions by using SYBR Green Supermix (Bio-Rad). The resulting signals were normalized for primer efficiencyby carrying out qPCR for each primer pair using Input DNA.Primer sequences are available upon request.

Quantitative PCR.RNA was purified from cells by using an RNeasyMini Kit (Qiagen) according to the manufacturer’s protocol.First-strand cDNA was synthesized by using SuperScript III re-verse transcriptase. Quantitative real-time PCR was performedby using FastStart Universal Probe master mix (Roche) anduniversal probe library probes (Roche) on a Roche Lightcycler480 II or Stratagene MxPro3005p. Samples were normalized byusing β2 microglobulin or GAPDH primer/probe sets. Primersequences are available upon request.

Chemical Compound Synthesis. (+)-JQ1 and (-)-JQ were synthe-sized according to published methods (Filippakopoulos et al.; ref.1) and iBET was synthesized according to published methods(Nicodeme et al.; ref. 2). Synthesis was provided by Alex Taylor,Victor Gehling, and Brian Albrecht.

Xenograft Efficacy Studies. Raji xenografts were established in 8-wk-old female NOD/SCID mice (Harlan) inoculated s.c. with 3 ×106 cells per mouse resuspended in 10% Matrigel. Two weekslater (average tumor volume 150 mm3), mice were assigned intotwo groups: 15 mice were treated with vehicle control (5:95DMSO:10% 2-Hydroxypropyl-β-cyclodextrin), and 15 mice weretreated with 30 mg/kg (+)-JQ1 by i.p. injection twice a day for28 d. Body weight and tumor volume were measured daily. Tu-mor volume was calculated from caliper measurements by usingthe following formula: W × H × L × 0.52. Mice were killed whentumor volume reached 2,000 mm3, when body weight decreased>20% of initial weight, or when the mice were in poor health asestablished in the IACUC protocol. Survival was plotted andanalyzed in GraphPad Prism software, and statistical significancewas calculated by using log-rank (Mantel-Cox) and Gehan–Breslow–Wilcoxon tests. MV4-11 xenografts were established innude mice injected with 10 × 106 cells per mouse. JQ1 was dosedi.p. and formulated as described above. Mice were divided into4 groups of 10 animals: vehicle control once a day; 50 mg/kg(+)-JQ1 once a day; 30 mg/kg (+)-JQ1 twice a day; and cytar-abine 100 mg/kg daily (5 d on, 2 d off). Treatment of mice withcytarabine at 100 mg/kg resulted in significant weight loss at day8 and, therefore, the dose needed to be decreased to 75 mg/kg.Mice were euthanized by CO2 exposure. Tumor volume datawere collected and analyzed as described above. All animal workwas performed according to approved IACUC protocols atVivisource or at WuXi.

MYC Expression in Raji Xenografts. Female NOD SCID mice(Harlan) were inoculated s.c. with 3 × 106 Raji cells. Tumors weregrown until they reached a size of 200 mm3 to 400 mm3 asmeasured by caliper. Mice were treated i.p. with either a singledose of 25 mg/kg (+)-JQ1 or vehicle. (+)-JQ1 was prepared ina solution of 5:95 DMSO:10% 2-Hydroxypropyl-β-cyclodextrin(Sigma) at a concentration of 5 mg/mL. At the indicated time

points after treatment, tumors were harvested. RNA was isolatedby using TRIzol extraction and processed for qPCR analysis.

Gene Expression Analysis. CEL files were processed by usingAffymetrix Expression Console program (http://media.affymetrix.com/support/downloads/manuals/expression_console_exon_level_quickstart_guide.pdf) and, specifically, the gene-level RMA al-gorithm using the core set of probesets. Data were exported fromExpression Console by using the function “Export Probe SetResults (pivot table) with Annotations to TXT”; version Huex-1_0-st-v2_na31_hg19 annotation files were downloaded from Af-fymetrix via the Expression Console program. Differential ex-pression scores were calculated separately for the BET inhibitorvs. the inactive enantiomer, at timepoints 4 and 8 h, and for twocell lines (LP-1 and Raji). The differential score for any givencell line and time point was

μactive − μinactive1þ σactive þ σinactive

:

Where μactive is the mean of the two log-expression values aftertreatment with the active compound, μinactive is the same for theinactive compound, σactive is the SD of the two log-expressionvalues after treatment with the active compound, and σinactive isthe same for the inactive compound. This score captures the foldchange (or, in log space, the difference) between the expressionvalues after the two compound treatments, while also adjustingfor noise: For measurements with a lot of noise (high SDs), thescore will have a lower magnitude.The four scores were averaged to create an overall score in-

dicating the differential expression across cell lines and time-points. Before GSEA analysis, the dataset was filtered to removetranscripts of genes for which there was a higher-expressedtranscript in the dataset.The complete list of genes along with their overall score was

used as input to GSEA [Subramanian et al. (3) and Mootha et al.(4)]. We focused on two of the five major MSIGDB collections:c2 (curated gene sets) and c3 (motif gene sets). We used thefollowing parameters in running GseaPreranked:

MYC shRNAs and Lentiviral Transduction. Target sequences for hu-man c-MYC (NM_002467.4) were obtained from Cellecta. Senseand antisense oligonucleotides (66 nt) containing sense, loop, andantisense sequences were obtained with 5′ phosphorylation fromIntegrated DNA Technologies. Oligonucleotides were annealedand ligated into a lentiviral shRNA vector based on pLKO.1obtained from Cellecta. In this vector, shRNA expression isdriven by the U6 promoter, and both a fluorescence marker andpuromycin acetyltransferase (separated by the T2A sequence) aredriven by the UbiC promoter. The target sequences of theshRNAs shown in the text are shRNA 1: 5′-GTCCTGAG-

GSEA Parameters

Norm meandivscoring_scheme weightedmake_sets TRUEmode Max_probegmx gseaftp.broadinstitute.org://pub/gsea/

gene_sets/c3.all.v3.0.symbols.gmtand gseaftp.broadinstitute.org://pub/gsea/gene_sets/c2.all.v3.0.symbols.gmt

include_only_symbols TRUEset_min 15nperm 1000rnd_seed timestamp

Mertz et al. www.pnas.org/cgi/content/short/1108190108 2 of 13

CAATCACCTATGA-3′; shRNA 2: 5′-CCTGAGCAATCACC-TATGAAC-3′; shRNA 3: 5′-CGACGAGAACAGTTGAAAC-AC-3′; and shRNA 4: 5′-GACGAGAACAGTTGAAACACA-3′.The sequence of the nontargeting control shRNA (NTC) is 5′-CGCTGAGTACTTCGAAATGTC-3′. Lentiviral production wascarried out according to publicly available protocols (http://www.broadinstitute.org/rnai/public/resources/protocols).For lentiviral transduction and assessment of growth inhibition,

LP-1 and MDA-MB-231 cells were transduced with virus ata multiplicity of infection (MOI) that gave 20–60% infection, asmeasured by determining the percentage of green cells at 24 hafter infection. At 4–5 d, the percentage of green cells was mea-

sured again, and the ratio of % green cells at 4 d to % green cellsat 1 d was calculated. In parallel, cells were transduced at a highMOI, selected with puromycin for 2 d, and RNA was prepared fordetermination of MYC mRNA expression as described above.

QuantiGene Assay. LP-1 cells were seeded in a 96-well plate andincubated in the presence of various concentrations of (+)-JQ1for 4 h. Relative mRNA levels were quantitated by usingQuantiGene 2.0 assay (Affymetrix) according to the manu-facturer’s recommendation. Signals were detected by using anEnvision plate reader (Perkin-Elme). Biological duplicates wereaveraged and normalized to vehicle (DMSO) control to calculatepercent MYC mRNA levels.

1. Filippakopoulos P, et al. (2010) Selective inhibition of BET bromodomains. Nature 468:1067e1073.

2. Nicodeme E, et al. (2010) Suppression of inflammation by a synthetic histone mimic.Nature 468:1119e1123.

3. SubramanianA, et al. (2005)Gene set enrichment analysis: A knowledge-basedapproach forinterpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102:15545e15550.

4. Mootha VK, et al. (2003) PGC-1alpha-responsive genes involved in oxidative phosphor-ylation are coordinately downregulated in human diabetes. Nat Genet 34:267e273.

DMSO

Eve

nts

PI

Viable: 96%G1: 45%S: 44%G2: 10%

sub-G1: 4%

(+)-JQ1

Eve

nts

PI

Viable: 36%G1: 84%S: 7%G2: 9%

sub-G1: 64%

Sub-G172%

(+)-JQ1 [5 µM]

Eve

nts

PI

Sub-G12.3%

(-)-JQ1 [5 µM]

Eve

nts

PI

A B

DMSO

(+)-JQ1

(-)-JQ1t0

2 h 4 h 6 h 8 h 16 h 24 h

G1: 33%S: 59%

G1: 59%

S: 37%

G1: 37%S: 61%

G1: 56%S: 28%

G1: 67%

S: 20%

G1: 55%S: 34%

C

Fig. S1. Cell cycle arrest and apoptosis induced by BET inhibition. (A) Cell cycle profile of Raji cells after treatment with DMSO or (+)-JQ1 [625 nM] for 72 hours.(B) Cell cycle profile of Raji cells after treatment with 5 μM (-)-JQ1 or (+)-JQ1 for 72 h. Data are representative of three independent experiments. (C) HMCLAMO-1 cells were synchronized by using a double thymidine block. The cells were released into DMSO or (-)-JQ1 or (+)-JQ1 at [625 nM]. Cells were fixed forFACS analysis at the indicated time points (h, hours). Cell cycle distribution was obtained with ModFit XL software. X-axis, PI; Y-axis, Events.

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Rel

ativ

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YC

expr

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Raji D

MS

O

Raji 546

Raji 565

Raji 417

LP1 DM

SO

LP1 546

LP1 565

LP1 417(+)-

JQ1

(-)-

JQ1

I-BET

DM

SO

Raji

(+)-

JQ1

(-)-

JQ1

I-BET

DM

SO

LP-1

0

0.2

0.4

0.6

0.8

1

1.2

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1.6

+ +- -

8 h4 h

AMO-1

Rel

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expressio

n

(+)-JQ1 + +- -

8 h4 h

+ +- -

8 h4 h

LP-1 Raji

A B

C

0.001 0.01 0.1 1 10

-20

0

20

40

60

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MYC mRNA

G1 increase

Apoptosis

(+)-JQ1 concentration (µM)

Percen

tag

e

0

1

2

3

4

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6

7

8

Untr 12 MRPL1 -269

DCTD -1654

IREB2 -619

Control(+)-JQ1

Bin

ding

eve

nts

BRD4 binding

P1 P2-200 bp

IgH

1395 bp

(+)-JQ1 +-

RSF1

H3K4me3

Beads

BRD3

BRD4

D E

Fig. S2. MYC suppression by BET inhibition. (A) Comparison of BET inhibitor-induced MYC suppression, G1 arrest, and apoptosis. LP-1 cells were exposed to theindicated concentrations of (+)-JQ1 for 4 h (mRNA expression) or 4 d (G1 arrest and apoptosis). mRNA expression was measured by using a Quantigene assay,and values were normalized to the control condition (mean ± standard deviation, n = 2). G1 arrest was measured by flow cytometric analysis of DNA contentand is expressed as the difference between the percentage of G1 cells at the indicated concentration and the percentage of G1 cells in the control condition(mean ± standard deviation, n = 6). Apoptosis was measured by using annexin V-PI staining. Values represent the total of early (annexin V positive, PI negative)and late (annexin V positive, PI positive) apoptosis (mean ± standard deviation, n = 3). (B) Raji and LP-1 cells were treated for 4 h with the indicated compoundsat a dose of [2.5 μM]. RNA from treated cells was used for qPCR analyses of MYC expression. Expression is shown relative to DMSO-treated cells. (C) qPCRanalysis of MYC expression in AMO-1, LP-1, and Raji cell lines were treated with GI90 doses of (+)-JQ1 or DMSO for 4 or 8 h (AMO-1 GI90 = [625 nM], LP-1 GI90 =[250 nM] and Raji GI90 = [250 nM]). (D) Chromatin immunoprecipitation from Raji cells treated with DMSO (-) or (+)-JQ1 (+) [500 nM] for 4 h using the indicatedantibodies. PCR was performed by using a primer set corresponding to the region indicated (gray box). The positions of P1 and P2 promoters and IgHtranslocation are indicated. (E) Chromatin immunoprecipitation from LP-1 cells treated with DMSO (-) or (+)-JQ1 [500 nM] for 4 h by using a BRD4 antibody.(+)-JQ1 treatment results in a loss of BRD4 binding to the MRPL1, DCTD, and IREB loci, but does not affect BRD4 binding to a gene desert of chromosome 12(Untr 12).

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MYC

Vin

(+)-JQ1 +-

Raji HL60 MV4-11 HeLa

+- +- +-

LP-1 AU565

+-

MDA

MB-231

+-+-

TranslocationAmplification

Non re-arranged

Viral insertion

+- (+)-JQ1

Raji

LP

-1

HL

60

AU

565

MD

A-

MB

-231

MV

4-11

He

La

100

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20

0

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Per

cent

MY

C expressio

n

+-+-+-+-+-+-

A

B

Fig. S3. Suppression of MYC mRNA and protein by BET inhibition in multiple cell lines. MYC expression was analyzed in a panel of cell lines treated with(+)-JQ1 or (-)-JQ1 for 4 h with a dose of [2.5 μM]. Cells were examined by qPCR (A) or Western blotting (B) for MYC expression. mRNA expression is relative to(-)-JQ1 treated samples for each cell line. (Vin, vinculin).

MYCVIN

(+)-JQ1 (-)-JQ1

- + - +Dox

B OFF DOX-MYC

ON DOX+MYC

100

80

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40

20

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MYC

BC

L-X

L

CD

C25

A

CC

ND

2

CD

KN

2A

E2F

4

TER

T

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ativ

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-MYC

ON DOX+MYC

25

20

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10

5

0

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35

Fold

indu

ctio

n

p21 APAF1 FOS SERTAD1

A

D

02468

101214161820

1

-MYC

+MYC

DOX +-

% A

popt

osis

Fig. S4. MYC neutralizes the deregulation of select genes upon BET inhibition. (A) LP-1 cells containing an inducible MYC construct (LP1-iMYC) were grown inthe presence of (-)-JQ1 or (+)-JQ1 at [125 nM] and were treated with or without doxycycline. Western blot analysis of MYC and vinculin were performed. (B andC) mRNA expression (qPCR) of the indicated genes after incubation with (+)-JQ1 for 4 h in LP-1 cells in the absence (MYC off) or the presence (MYC on) ofdoxycycline. Relative gene expression was calculated by comparing cells on-doxycycline/(+)-JQ1 to on doxycycline/(-)-JQ1, and off doxycycline/(+)-JQ1 to offdoxycycline/(-)-JQ1. (mean ± standard deviation, n = 2). (D) LP1-iMYC cells were grown in the presence or absence of doxycycline (1 μM) for 3 d. Apoptosis wasmeasured as the percentage of cells with sub-G1 DNA content. (mean ± standard deviation, n = 3).

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0

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)

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od

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ht ch

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)C D

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ssio

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(+)-JQ1+ +--EL4 RAW 264.7

Fig. S5. BET-bromodomain inhibition decreases tumor load in vivo and is well tolerated in mice. (A) The fraction of human CD45+CD19+ cells in the bonemarrow of NOD/SCID mice inoculated i.v. with Raji cells measured by FACS analysis at day 15. (+)-JQ1 was administered i.p. at 50 mg/kg, once per day, starting48 hours after Raji cell inoculation. (+)-JQ1, nine animals per group; vehicle control, eight animals per group. (B) MYC mRNA expression measured by qPCR inRaji cells extracted from bone marrow 3 h after the last dose of (+)-JQ1. *P < 0.005; **P < 0.001 in a two-tailed Student t test. (C) Change in body weight overtime in the Raji subcutaneous xenograft efficacy study. (D) Change in body weight over time in the MV4-11 subcutaneous xenograft efficacy study. (E) Themurine cell lines EL4 and RAW 264.7 were exposed to (+)-JQ1 or (-)-JQ1 (2.5 μM) for 4 h. RNA was prepared and Myc expression was measured by qPCR.Expression was calculated relative to (-)-JQ1.

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0

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NTC sh 1 sh 2 sh 3 sh 4

% G

FP 4

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% G

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Viability0

20406080

100120140

Rel

ativ

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YC

Nex

pres

sion

(%)

- +(+)-JQ1

Neuroblastoma Lung Liver

SKNDZ NCI-H69 BE(2)C CHP212 SNU182

- + - + - + - +

Fig. S6. (A) Suppression of MYC in cells phenotypically affected by BET inhibition. Cells were treated for 4 h with a dose of 2.5 μM (+)-JQ1 or DMSO, and MYCtranscript levels were analyzed by qPCR. For each cell line, expression is relative to DMSO treated cells (-). (B) Knockdown of MYC mRNA does not reduce theviability of MDA-MB-231 cells. (Upper) MYC expression after shRNA transduction in MDA-MB-231 cells. Cells were grown under antibiotic selection for 3 d, andRNA was prepared for analysis of MYC expression by qPCR. Expression was calculated relative to the nontargeting control (NTC) shRNA (mean ± standarddeviation, n = 2). (Lower) After 24 h, the percentage of green cells was determined by flow cytometric analysis. Cells were grown without selection for anadditional 4 d, and the percentage of green cells was measured. Values represent the depletion of shRNA-containing cells relative to uninfected cells (mean ±standard deviation, n = 2). (C) BET inhibitors suppress MYCN in the context of genomic amplification. MYCN expression was analyzed in a panel of MYCN-amplified cell lines treated with the BET inhibitor (+)-JQ1 or DMSO (-) for 4 h with a dose of 2.5 μM. RNA from treated cells was used for qPCR of MYCN.Expression is shown relative to DMSO-treated cells.

Mertz et al. www.pnas.org/cgi/content/short/1108190108 7 of 13

Table S1. Top 20 most differentially down-regulated and up-regulated genes upon BET inhibitor treatment

Probe set mRNA accession Gene symbol

Mean logexpression

LP1inactive

enantiomer

Mean logexpression

LP1 with BETinhibitor

Mean logexpression

Rajiinactive

enantiomer

Mean logexpressionRaji with

BETinhibitor

Logtwofoldchange

Differentialexpression

score

Down-regulation3115504 NM_002467 MYC 9.5 7.43 8.45 7.05 −1.74 −1.482672096 NM_001295 CCR1 7.1 3.54 2.9 2.55 −1.95 −1.363648391 NM_001192 TNFRSF17 8.95 7.06 4.69 3 −1.79 −1.193389878 NM_138775 ALKBH8 7.39 5.56 7.32 6.04 −1.55 −1.172403446 NM_001164721 PTAFR 4.07 3.35 7.99 4.97 −1.87 −1.122773958 NM_001565 CXCL10 8.09 6.64 4.96 3.39 −1.51 −1.022860178 NM_005582 CD180 5.03 4.35 8.21 6.46 −1.22 −0.973768412 NM_001174166 SLC16A6 6.61 4.78 8.94 8.09 −1.34 −0.932412988 NM_023077 C1orf163 7.22 5.63 7.28 6.28 −1.29 −0.922977471 NM_182503 ADAT2 7.48 5.9 8.62 7.79 −1.21 −0.892372781 NM_002922 RGS1 6.23 4.24 5.08 4.43 −1.32 −0.892378180 NM_014388 C1orf107 7.03 5.61 6.99 6.09 −1.16 −0.872363202 NM_021181 SLAMF7 7.29 6.14 4.71 3.81 −1.03 −0.873244061 NR_026693 ZNF487 6.05 4.33 5.63 4.82 −1.27 −0.873398145 NM_020228 PRDM10 5.6 4.9 6.55 5.13 −1.07 −0.862754673 NM_181726 ANKRD37 8.92 7.03 5.51 5.59 −0.9 −0.833326826 NM_014344 FJX1 8.17 6.32 5.1 5.03 −0.96 −0.823908149 NM_183047 ZMYND8 5.52 4.52 6.63 5.68 −0.98 −0.812440354 NM_001778 CD48 9.5 8.54 9.54 8.5 −1 −0.812926802 NM_001130173 MYB 9.13 8.12 7.76 6.73 −1.02 −0.79

Up-regulation2434031 NM_001024599 HIST2H2BF 4.83 8.27 3.36 4.92 2.5 1.712946681 NM_021058 HIST1H2BJ 6.03 8.44 3.62 5.91 2.35 1.492900143 NM_012367 OR2B6 3.48 6.58 2.25 4.89 2.87 1.442366422 NM_001677 ATP1B1 4.88 7.51 4.89 5.97 1.86 1.413723348 NM_006460 HEXIM1 7.1 8.35 6.31 8.23 1.58 1.343971806 NR_027783 SAT1 5.46 7.6 7.72 8.95 1.68 1.293454662 NM_030809 CSRNP2 6.6 8.09 6.12 7.66 1.51 1.162899756 NM_021064 HIST1H2AG 7.05 9.47 6.17 6.99 1.62 1.152899176 NM_138720 HIST1H2BD 6.69 9.24 6.12 7.88 2.15 1.143718555 NM_144975 SLFN5 5.01 6.33 3.91 5.74 1.58 1.133376155 NM_001081491 NXF1 8.94 10.4 9.07 10.2 1.29 1.122434124 NM_003528 HIST2H2BE 6.92 9.24 5.18 6.35 1.74 1.073387259 NM_144665 SESN3 7.74 8.52 6.55 8.35 1.29 1.072426840 NM_001142550 WDR47 5.09 6.85 5.91 7.29 1.57 1.062356115 NM_006472 TXNIP 7.3 8.83 8.69 9.82 1.33 1.063708704 NM_000937 POLR2A 8.25 9.36 8.31 9.6 1.2 1.032946714 NM_080593 HIST1H2BK 7.8 8.77 4.47 6.8 1.65 1.023019565 NM_182597 C7orf53 3.27 4.36 3.12 4.76 1.37 1.023453837 NM_006009 TUBA1A 7.39 8.46 7.97 9.13 1.11 0.983209384 NM_013390 TMEM2 5.51 7.57 3.87 4.4 1.29 0.98

Expression changes across LP1 and Raji cell lines were obtained as described in SI Methods.

Mertz et al. www.pnas.org/cgi/content/short/1108190108 8 of 13

Table S2. Top 20 down-regulated and up-regulated gene sets for GSEA c2 (curated gene sets) analysis

Gene set name Description Size ES NES FDR q value Rank

Down-regulatedSCHLOSSER_MYC_TARGETS_

AND_SERUM_RESPONSE_UP

Genes up-regulated in B493-6 cells (Blymphocytes) by MYC alone or in combinationwith serum but not by serum alone

46 −0.69693 −2.36288 0 1

SCHLOSSER_MYC_TARGETS_AND_SERUM_RESPONSE_DN

Genes down-regulated in B493-6 cells (Blymphocytes) by MYC in combination withserum but not affected by serum alone; theyare also up-regulated by MYC alone

44 −0.70889 −2.33524 0 2

HELLER_HDAC_TARGETS_SILENCED_BY_METHYLATION_DN

Genes down-regulated in multiple myeloma(MM) cell lines treated with both decitabine TSA

235 −0.62631 −2.73672 0 3

HELLER_HDAC_TARGETS_DN Genes down-regulated in at least one of threemultiple myeloma (MM) cell lines by TSA

240 −0.60531 −2.61576 0 4

SCHUHMACHER_MYC_TARGETS_UP

Genes up-regulated in P493-6 cells (Burkitt’slymphoma) induced to express MYC

63 −0.63867 −2.28145 1.67E-04 5

REACTOME_METABOLISM_OF_RNA

Genes involved in Metabolism of RNA 87 −0.60144 −2.26806 2.77E-04 6

VANTVEER_BREAST_CANCER_BRCA1_UP

Up-regulated genes from the optimal set of100 markers discriminating ER(-) breastcancer tumors by BRCA1 mutation status

29 −0.70215 −2.19 0.001058 7

SCHLOSSER_MYC_TARGETS_REPRESSED_BY_SERUM

Genes up-regulated in B493-6 cells (Blymphocytes) by MYC and down-regulatedby the combination of MYC and serum

149 −0.53249 −2.15476 0.001341 8

REACTOME_SNRNP_ASSEMBLY

Genes involved in snRNP assembly 43 −0.65648 −2.1493 0.001192 9

MANALO_HYPOXIA_DN Genes down-regulated in response to bothhypoxia and overexpression of an active formof HIF1A

261 −0.48772 −2.14522 0.001073 10

KEGG_PRIMARY_IMMUNODEFICIENCY

Primary immunodeficiency 35 −0.67613 −2.14332 9.75E-04 11

YANG_BREAST_CANCER_ESR1_LASER_DN

Genes down-regulated in laser microdissected(LCM) samples of early primary breast tumorsexpressing ESR1 vs. the ESR1 negative ones

35 −0.64864 −2.07892 0.001798 12

BERTUCCI_MEDULLARY_VS_DUCTAL_BREAST_CANCER_UP

Genes up-regulated in medullary breast cancer(MBC) relative to ductal breast cancer (DBD)

178 −0.50264 −2.07835 0.001659 13

BILD_MYC_ONCOGENIC_SIGNATURE

Genes selected in supervised analyses todiscriminate cells expressing c-Myc fromcontrol cells expressing GFP

173 −0.50228 −2.07447 0.001718 14

WALLACE_PROSTATE_CANCER_RACE_UP

Genes up-regulated in prostate cancer samplesfrom African-American patients compared withthose from the European-American patients

264 −0.4619 −2.02806 0.004213 15

WILENSKY_RESPONSE_TO_DARAPLADIB

Atherosclerotic process genes whose coronaryexpression changed after darapladib treatment

26 −0.66815 −2.00107 0.005765 16

JAIN_NFKB_SIGNALING Genes abnormally regulated in response to CD40Land IL4 stimulation of B lymphocytes frompatients with a hypomorphic mutation of IKBKG

72 −0.54424 −1.99492 0.006058 17

REACTOME_CYTOSOLIC_TRNA_AMINOACYLATION

Genes involved in Cytosolic tRNA aminoacylation 23 −0.68141 −1.97872 0.006833 18

MORI_PLASMA_CELL_DN Down-regulated genes in the B lymphocytedevelopmental signature, based on expressionprofiling of lymphomas from the Emu-myctransgenic mice: plasma cell

17 −0.72819 −1.95117 0.009451 19

SHAFFER_IRF4_TARGETS_IN_ACTIVATED_DENDRITIC_CELL

IRF4 target genes up-regulated in plasmacytoiddendritic cells compared with monocytes

62 −0.54406 −1.91516 0.014964 20

Up-regulatedHELLER_HDAC_TARGETS_

SILENCED_BY_METHYLATION_UP

Genes up-regulated in multiple myeloma (MM)cell lines treated with both decitabine TSA

366 0.59171 2.552606 0 1

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Table S2. Cont.

Gene set name Description Size ES NES FDR q value Rank

REACTOME_RNA_POLYMERASE_I_PROMOTER_OPENING

Genes involved in RNA Polymerase I PromoterOpening

45 0.782485 2.520709 0 2

REACTOME_PACKAGING_OF_TELOMERE_ENDS

Genes involved in Packaging Of Telomere Ends 39 0.807121 2.481099 0 3

ZHONG_RESPONSE_TO_AZACITIDINE_AND_TSA_UP

Genes up-regulated in 3 of 4 NSCLC cell lines(nonsmall cell lung cancer) aftertreatment with azacitidine and TSA

169 0.603664 2.434109 0 4

DACOSTA_UV_RESPONSE_VIA_ERCC3_COMMON_UP

Common up-regulated transcripts in fibroblastsexpressing either XP/CS or TDD mutant formsof ERCC3, after UVC irradiation

53 0.715886 2.384411 0 5

HELLER_HDAC_TARGETS_UP Genes up-regulated in at least one of threemultiple myeloma (MM) cell lines by TSA

246 0.569763 2.370475 0 6

REACTOME_RNA_POLYMERASE_I_PROMOTER_CLEARANCE

Genes involved in RNA PolymerasePromoterClearance

67 0.661081 2.354096 0 7

KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS

Systemic lupus erythematosus 110 0.61959 2.331192 0 8

PICCALUGA_ANGIOIMMUNOBLASTIC_LYMPHOMA_DN

Down-regulated genes in angioimmunoblasticlymphoma (AILT) compared with normal Tlymphocytes

119 0.595525 2.270817 0 9

REACTOME_TELOMERE_MAINTENANCE

Genes involved in Telomere Maintenance 65 0.652077 2.268739 1.10E-04 10

DACOSTA_UV_RESPONSE_VIA_ERCC3_UP

Genes up-regulated in fibroblasts expressingmutant forms of ERCC3 after UV irradiation

289 0.495406 2.112918 0.001122 11

AMIT_EGF_RESPONSE_40_HELA

Genes whose expression peaked at 40 min afterstimulation of HeLa cells with EGF

40 0.657129 2.084719 0.001407 12

CHIBA_RESPONSE_TO_TSA Genes up-regulated in more than one of severalhuman hepatoma cell lines by TSA

37 0.659875 2.083599 0.001299 13

LIU_CMYB_TARGETS_UP Genes up-regulated in MCF-7 cells (breast cancer)by overexpression of CMYB off adenovirus vector

150 0.523566 2.059998 0.001773 14

SESTO_RESPONSE_TO_UV_C0 Genes changed in primary keratinocytes by UVBirradiation

104 0.544836 2.046169 0.002332 15

DAZARD_RESPONSE_TO_UV_SCC_UP

Genes up-regulated in SCC12B2 cells (squamouscell carcinoma) by UV-B irradiation

80 0.567033 2.022694 0.003097 16

BIOCARTA_EGF_PATHWAY EGF Signaling Pathway 30 0.678379 2.001748 0.004572 17SWEET_KRAS_TARGETS_DN Genes down-regulated in A549 cells (lung cancer)

after RNAi knockdown of activated KRAS26 0.693547 2.001021 0.004512 18

DACOSTA_UV_RESPONSE_VIA_ERCC3_XPCS_UP

Genes exclusively up-regulated in fibroblastsexpressing the XP/CS mutant form of ERCC3after high dose UVC irradiation

16 0.760104 1.99926 0.004274 19

AMIT_SERUM_RESPONSE_40_MCF10A

Genes whose expression peaked at 40 minafter stimulation of MCF10A cells with serum

32 0.665377 1.994091 0.00451 20

Parameters for GSEA are indicated in SI Methods. ES, enrichment score. NES, normalized enrichment score. FDR, false discovery rate.

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Table S3. Top 20 down-regulated and up-regulated gene sets for GSEA c3 (motif gene sets) analysis

Gene set name Description Size ES NES FDR q-value Rank

Down-regulatedV$MYCMAX_01 Genes with promoter regions [-2kb,2kb] around

transcription start site containing the motifNNACCACGTGGTNN which matches annotationfor MYC: v-myc myelocytomatosis viral oncogenehomolog (avian) MAX: MYC associated factor X

182 −0.42194 −1.77581 0.079094 1

GTCAGGA,MIR-378 Targets of MicroRNA GTCAGGA,MIR-378 41 −0.4989 −1.65604 0.164508 2V$IRF_Q6 Genes with promoter regions [-2kb,2kb]

around transcription start site containingthe motif BNCRSTTTCANTTYY whichmatches annotation for IRF1: IFNregulatory factor 1

167 −0.38082 −1.58381 0.235204 3

RRCCGTTA_UNKNOWN Genes with promoter regions [-2kb,2kb]around transcription start site containingmotif RRCCGTTA.Motif does not match any known transcriptionfactor

52 −0.44918 −1.5588 0.220582 4

V$STAT3_01 Genes with promoter regions [-2 kb, 2 kb]around transcription start sitecontaining the motif NGNNATTTCCSGGAARTGNNNwhich matches annotation for STAT3: signaltransducer and activator of transcription 3(acute-phase response factor)

16 −0.57277 −1.52693 0.241739 5

KTGGYRSGAA_UNKNOWN

Genes with promoter regions [-2kb,2kb]around transcription start site containing motifKTGGYRSGAA. Motif doesnot match any known transcription factor

54 −0.42682 −1.50386 0.254988 6

V$NMYC_01 Genes with promoter regions [-2kb,2kb] aroundtranscription start site containing the motifNNCCACGTGNNN which matches annotationfor MYCN: v-myc myelocytomatosis viral relatedoncogene, neuroblastoma derived (avian)

193 −0.34785 −1.48038 0.273182 7

AACYNNNNTTCCS_UNKNOWN

Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing motifAACYNNNNTTCCS. Motif does not match anyknown transcription factor

60 −0.40379 −1.44763 0.328743 8

CTCNANGTGNY_UNKNOWN

Genes with promoter regions [-2kb,2kb] aroundtranscription start site containing motifCTCNANGTGNY. Motif does not match anyknown transcription factor

71 −0.39112 −1.4251 0.362743 9

V$GNCF_01 Genes with promoter regions [-2kb,2kb]around transcription start site containingthe motif NTCAAGKTCAAGKTCANNwhich matches annotation for NR6A1:nuclear receptor subfamily 6, group A,member 1

55 −0.40776 −1.42352 0.33123 10

V$PPARG_01 Genes with promoter regions [-2kb,2kb]around transcription start site containingthe motif NNWGRGGTCAAAGGTCANNNNwhich matches annotation for PPARG: peroxisomeproliferative activated receptor, gamma

32 −0.45739 −1.4188 0.315147 11

V$E47_02 Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing the motifNNNMRCAGGTGTTMNN which matchesannotation for TCF3: transcription factor3 (E2A Ig enhancer binding factors E12/E47)

182 −0.33505 −1.41843 0.2899 12

RNTCANNRNNYNATTW_UNKNOWN

Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing motifRNTCANNRNNYNATTW. Motifdoes not match any known transcriptionfactor

47 −0.41591 −1.41201 0.284802 13

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Table S3. Cont.

Gene set name Description Size ES NES FDR q-value Rank

V$ISRE_01 Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing the motifCAGTTTCWCTTTYCC whichmatches annotation for STAT1: signal transducerand activator of transcription 1, 91kDa STAT2:signal transducer and activatorof transcription 2, 113kDa

165 −0.33179 −1.36516 0.40618 14

V$NFKB_Q6 Genes with promoter regions [-2kb,2kb] aroundtranscription start site containing motifNGGGGAMTTTCCNN.

191 −0.31496 −1.34326 0.460806 15

V$AP1_Q4 Genes with promoter regions [-2 kb, 2 kb]around transcription start site containing the motifRGTGACTMANN which matches annotation forJUN: jun oncogene

209 −0.31272 −1.3422 0.436019 16

V$USF_C Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing motif NCACGTGN.

203 −0.31119 −1.3388 0.422637 17

V$MAX_01 Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing the motifNNANCACGTGNTNN whichmatches annotation for MAX: MYC associatedfactor X

186 −0.3115 −1.33374 0.417266 18

RGAGGAARY_V$PU1_Q6 Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing the motifRGAGGAARY which matches annotation forSPI1: spleen focus forming virus (SFFV) proviralintegration oncogene spi1

357 −0.29346 −1.33078 0.405792 19

TTANWNANTGGM_UNKNOWN

Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing motifTTANWNANTGGM. Motif does not match anyknown transcription factor

46 −0.39072 −1.32713 0.398241 20

Up-regulatedKRCTCNNNNMANAGC_

UNKNOWNGenes with promoter regions [-2 kb, 2 kb] around

transcription start site containing motifKRCTCNNNNMANAGC. Motif does not matchany known transcription factor. (Includes manyhistone genes)

54 0.754246 2.55403 0 1

TTTNNANAGCYR_UNKNOWN

Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing motifTTTNNANAGCYR. Motif does not match anyknown transcription factor. (Includes manyhistone genes)

113 0.559811 2.105364 0 2

ACTTTAT,MIR-142–5P Targets of MicroRNA ACTTTAT,MIR-142–5P 240 0.471313 1.968661 0.004958 3GTACAGG,MIR-486 Targets of MicroRNA GTACAGG,MIR-486 47 0.554023 1.817974 0.031404 4V$NFY_C Genes with promoter regions [-2 kb, 2 kb] around

transcription start site containing motifNCTGATTGGYTASY. (Includes a few histone genes)

177 0.446387 1.784838 0.037659 5

GTGCAAT,MIR-25,MIR-32,MIR-92,MIR-363,MIR-367

Targets of MicroRNA GTGCAAT,MIR-25,MIR-32,MIR-92,MIR-363,MIR-367

249 0.421978 1.774541 0.036779 6

CAGTGTT,MIR-141,MIR-200A

Targets of MicroRNA CAGTGTT,MIR-141,MIR-200A 256 0.41672 1.75204 0.040492 7

ACACTGG,MIR-199A,MIR-199B

Targets of MicroRNA ACACTGG,MIR-199A,MIR-199B 132 0.440497 1.713703 0.059971 8

CAGTATT,MIR-200B,MIR-200C,MIR-429

Targets of MicroRNA CAGTATT,MIR-200B,MIR-200C,MIR-429

375 0.389258 1.703134 0.06069 9

V$NKX22_01 Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing the motifTTAAGTRSTT which matches annotation forNKX2-2: NK2 transcription factor related,locus 2 (Drosophila)

136 0.430585 1.66127 0.085563 10

V$NFY_Q6 Genes with promoter regions [-2 kb, 2 kb] aroundtranscription start site containing motifTRRCCAATSRN.

185 0.410697 1.644636 0.095779 11

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Table S3. Cont.

Gene set name Description Size ES NES FDR q-value Rank

GGCAGCT,MIR-22 Targets of MicroRNA GGCAGCT,MIR-22 193 0.398895 1.615549 0.117168 12V$SP1_Q6 Genes with promoter regions [-2 kb, 2 kb] around

transcription start site containing the motifNGGGGGCGGGGYN which matches annotationfor SP1: Sp1 transcription factor

172 0.400724 1.611233 0.113358 13

GTAAACC,MIR-299–5P Targets of MicroRNA GTAAACC,MIR-299–5P 42 0.492953 1.601155 0.115242 14GTTATAT,MIR-410 Targets of MicroRNA GTTATAT,MIR-410 79 0.439599 1.582601 0.130842 15AGTCAGC,MIR-345 Targets of MicroRNA AGTCAGC,MIR-345 47 0.482915 1.581437 0.124393 16GCACTTT,MIR-17–5P,

MIR-20A,MIR-106A,MIR-106B,MIR-20B,MIR-519D

Targets of MicroRNA GCACTTT,MIR-17–5P,MIR-20A,MIR-106A,MIR-106B,MIR-20B,MIR-519D

479 0.352566 1.579857 0.118471 17

GTATTAT,MIR-369–3P Targets of MicroRNA GTATTAT,MIR-369–3P 156 0.398601 1.57092 0.122178 18GCATTTG,MIR-105 Targets of MicroRNA GCATTTG,MIR-105 136 0.397983 1.569191 0.118045 19AGCATTA,MIR-155 Targets of MicroRNA AGCATTA,MIR-155 112 0.415363 1.567574 0.114151 20

Parameters for GSEA are indicated in SI Methods. ES, enrichment score. NES, normalized enrichment score. FDR, false discovery rate.

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