Supporting InformationLee et al. 10.1073/pnas.1205132109SI Materials and MethodsGeneration of HCT116 Cell Lines Harboring Endogenous STAT3 K685RMutation. To mutate endogenous STAT3 acetylation, a 1.1-kbfragment from intron 19 to intron 21 of the STAT3 locus con-taining the sequences of exon 20 and exon 21 was amplified fromgenomic DNA of HCT116 cells using primers 5′-gggaaag(d)Utgtaaccaagtcccctgctc-3′ and 5′-ggagaca(d)Ugtcaccccaacaaaagc-act-3′. The coding sequence for K685 was then mutated fromAAG to AGG (Arg) by site-directed mutagenesis using primers5′-GGTATTGTCGGCCAGAGAG-3′ and 5′-CTCTGGCCGA-CAATACCTTCCGAATGCCTCCTCCTTG-3′. This mutatedfragment was used as the right homologous arm. Another 1.2-kbfragment was also amplified from genomic DNA as the left ho-mologous arm using primers 5′-GGTCCCA(d)UGCTCCACA-GAGAGTGCATGA-3′ and 5′-GGCATAG(d)UCACTGAGG-ACAGCTGCAGAG-3′. The two fragments were cloned into apAAV-Neo-USER-LoxP vector by USER treatment. The tar-geting AAV viruses were packaged in 293T cells by transfectingequal amounts of the targeting vector, pHelper and pRC plas-mids. Viruses were harvested 72 h after transfection. HCT116cells were infected with the targeting viruses and selected with0.4 mg/mL genectin for 20 d. The genectin-resistant clones werethen screened for homologous recombination by genomic PCRwith primers derived from the neomycin resistance gene (5′-GTTGTGCCCAGTCATAGCCG-3′) and the upstream regionof the left homologous arm (5′- TTGGAACGAAGGGTAGG-TTG-3′). After the first allele was targeted, the neomycin re-sistance gene was excised by Cre recombinase and targetedclones were retargeted to obtain homozygous knock-in cells.Targeted clones were sequenced to validate the presence of theintended mutation. HCT116 cells were maintained in McCoy 5Amedia plus 10% (vol/vol) FBS.

Reagents. Polyclonal antibodies recognizing STAT3 (C-20 and C-20x), estrogen receptor-α (ERα) (F-10) and siRNA against hu-man STAT3 were from Santa Cruz Biotechnology; phospho-STAT3 (Y705) and acetyl-STAT3 (K685) antibodies were fromCell Signaling Technology; antibody specific to DNA methyl-transferase 1 (Dnmt1) was purchased from Stressgen. Antibodyto GFP that recognizes YFP was obtained from Rockland andantibody to 5′-methylcytosine from EMD4Biosciences.TSA and tamoxifen were purchased from Sigma-Aldrich and

used at 400 ng/mL and 2 μM, respectively. Ethanol was used asa solvent control for Trichostatin and tamoxifen. Resveratrol andanacardic acid were obtained from Cayman and dissolved inacetone or DMSO. Recombinant human IL-6 was obtained fromPeproTech and used at 20 ng/mL.The constructs pcDNA5/FRT/TO-YFP-STAT3 wild-type and

pRC/CMV-FLAG-STAT3 have been described (1, 2). STAT3K685R mutant was generated by site-directed mutagenesis usingYFP-STAT3 wild-type as template.

In Vivo Xenograft Tumor Models. To establish human tumor xen-ografts in mice, pcDNA5/FRT-TO/YFP-STAT3 wild-type orK685R plasmids were introduced into A2058 human melanomacells using Lipofectamine 2000. Twenty-four hours after trans-fection, YFP+ cells were enriched by Flow cytometry. Next, 1 ×106 cells were mixed with Matrigel enriched with growth factorsat 1:1 ratio then implanted into the flank of either athymic nudemice or NSG (NOD Scid-γ; NOD.Cg-Prkdcscidl2rgtm1Wjl/SzJ)mice. Tumor growth was monitored and recorded for the indicatedtime. To establish mouse tumor xenograft in mice, pcDNA5/FRT-

TO/YFP-STAT3 wild-type or K685R plasmids were introducedinto Stat3-deficient mouse embryonic fibroblasts (MEFs) usingLipofectamine 2000. Twenty-four hours after transfection, YFP+

cells were enriched by Flow cytometry. Next, 1 × 106 cells weremixed with Matrigel enriched with growth factors at 1:1 ratio thenimplanted into the flank of NSG mice.To test antitumor effects of resveratrol, A2058 cells were

subcutaneously implanted into immune-deficient NSG mice.When the tumor was palpable, mice were given 50 mg/kgresveratrol in 100 μL Neobee oil (Spectrum Chemicals) by in-tratumoral injection twice a week. For the control group, thesolvent DMSO was used in Neobee oil as a control. To establishMDA-MB468 triple-negative breast tumor models, 1 × 106 cellswere implanted subcutaneously into the flank of athymic nudemice. When tumor volume reached ∼150 mm3, mice were giventhe solvent control (combination of acetone and ethanol,0.001%), resveratrol (25 mg/kg), tamoxifen (1 mg/kg), or a com-bination of both in Neobee oil, three times a week for the in-dicated time periods by intraperitoneal injection.

Methylated DNA Immunoprecipitation. Briefly, genomic DNA (20μg) was diluted in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mMEDTA) and sheared to between 300 and 1,000-bp fragmentsusing a sonicator (Fisher Scientific). Four micrograms of soni-cated DNA was diluted in 450 μL of TE buffer and denatured for10 min at 95 °C and immediately placed on ice. Some of theDNA was saved as input. Sheared DNA was incubated with 5 μgof anti-5′ methylcytosine antibodies in IP buffer (10 mM Na-Phosphate, pH 7.0, 0.14 M NaCl, 0.05% Triton-X100) for 18 hbefore adding 40 μL of protein G agarose (prewashed in 0.1%BSA-PBS and equilibrated in IP buffer). Samples were washedthree times using IP buffer then incubated with proteinase K for3 h at 50 °C. Methylated DNA was purified using phenol/chlo-roform/isoamyl alcohol and ethanol precipitated. DNA was re-suspended in 60 μL TE and 2 μL of input and methylated DNAwas used for PCR. All PCR primers amplifying methylated re-gions in the promoter were obtained from Qiagen. Data werenormalized to input value. For the antibody control, shearedDNA was incubated with preimmune serum (PIS) and processedat the same time.

ChIP Assay. Cells were seeded in 150-mm plates at the density of1 × 106. Forty-eight hours after transfection, formaldehyde wasadded into culture media to a final concentration of 1% andincubated at room temperature for 10 min. Briefly, SDS lysisbuffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.0 and PMSF)was added to the cell pellet to isolate chromatin, and the lysateswere sonicated to shear DNA to an average length of 200–500bp. Input was prepared by treating aliquots of chromatin withproteinase K, heated at 65 °C for 6 h for decrosslinking, followedby ethanol precipitation. Pellets were resuspended, and the re-sulting DNA was quantified on a NanoDrop spectrophotometer.After overnight incubation with primary antibody at 4 °C, proteinA agarose beads were added to isolate immune complexes.Complexes were washed and eluted from the beads with elutionbuffer (1% SDS and 0.1M NaHCO3). Crosslinks were reversedby incubating at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitativereal-time PCR reactions were performed with ChIP-boundand input DNA. The resulting signals were normalized as(DNA amount in antibody-specific IP − DNA in PIS IP)/(DNAin input).

Lee et al. www.pnas.org/cgi/content/short/1205132109 1 of 4

Primers used in the study are:

CDKN2A: 5′-CACATTCGCTAAGTGCTCGGAGTT-3′ (for-ward), 5′-TCCTCTTTCTTCCTCCGGTGCT-3′ (reverse);

STAT1: 5′-AGTGAATGAGTCTCGAGGATCCGA-3′ (for-ward), 5′-AGACCTGAGACTGGGCAATTTACA-3′(reverse);

DLEC1: 5′-CTCCTTCTTTGAAATCTCTGCTTGG-3′ (for-ward), 5′-TTTAGTGGGTTAAGGCCAAGGTGG-3′(reverse);

PTPN6: 5′-TCTCTTGCAAGCATTGGCAAGGTC-3′ (forward),5′-TGCCAGGATTCAAACCCAGACAGT-3′ (reverse);

SOCS3: 5′-ACCCTCCGCGCTCAGCCTTT-3′ (forward), 5′-AGCGGAGCAGGGAGTCCAAGT-3′ (reverse).

1. Herrmann A, et al. (2007) Nucleocytoplasmic shuttling of persistently activated STAT3.J Cell Sci 120:3249–3261.

2. Lee H, et al. (2009) Persistently activated Stat3 maintains constitutive NF-kappaBactivity in tumors. Cancer Cell 15:283–293.

BA Tumor 1 Tumor 2 Tumor 1 Tumor 2 Tumor 3 Tumor 4

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Fig. S1. Acetylated STAT3 in malignant vs. nonmalignant human tissues. (A) Immunohistochemical (IHC) staining to measure acetylated STAT3 in normal vs.malignant human colon cancer tissues. (Upper) H&E staining of colon cancer tissue sections identifying normal vs. cancerous areas. A, adenomatous colontissue; C, cancerous colon tissue; H, hyperplastic colon tissue; N, the normal colon tissue area. (Lower) IHC staining of acetylated STAT3. (Scale bars, 100 μm.) (B)Acetylated STAT3 levels in tumor tissue sections of triple-negative breast cancer (TNBC) compared with matched normal breast tissues. (Scale bars, 100 μm.) (C)Real-time PCR quantifying mRNA levels of the indicated genes in MEF tumors expressing control, wild-type STAT3, or STAT3 K685R mutant.

WT WT KR WT WT KR STAT3TCM

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Fig. S2. Western blotting of immunoprecipitated STAT3 showing levels of endogenous STAT3 acetylation as well as phosphorylation in HCT116 human coloncancer cells, containing either wild-type STAT3 or STAT3 K685R in which the STAT3 acetylation site is mutated by targeted homologous recombination. TCM,tumor conditioned medium, used to further stimulate STAT3 activation in the tumor cells.

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Fig. S3. STAT3 acetylation is important for its interaction with DNMT1. (A) Western blotting of immunoprecipitated STAT3 using anti-STAT3 antibodiesshowing increased interaction of acetylated STAT3 with DNMT1. (B, Left) Acetylated STAT3-DNMT1 interaction in MCF7 tumor cells exposed to TCM. (Right)STAT3 acetylation levels in MCF7 cells treated with histone deacetylases (HDAC) inhibitor, Trichostatin (TSA), were analyzed by coimmunoprecipitation/Western blotting. (C) Western blotting of immunoprecipitated STAT3 showing a decrease in STAT3-DNMT1 interaction in MDA-MB468 human breast cancercells treated with acetyltransferase inhibitor anacardic acid for 18 h. (D) Immunofluorescent staining of acetylated STAT3 and DNMT1 in diverse human tumortissues. (Scale bars, 10 μm.)

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Fig. S4. Treatment of TNBC cells with resveratrol restores ERα expression and sensitizes cells to antiestrogen therapy by inhibiting STAT3 acetylation. (A, Left)RNA expression levels of ERα in MDA-MB231 TNBC cells treated with either solvent control or resveratrol. (Right) Western blotting showing protein expressionlevels of ERα and acetylated STAT3 in MDA-MB231 after treatment with either control or resveratrol. (B) Quantification of ERα promoter DNA methylationlevels in control- and resveratrol-treated MDA-MB231 cells by EpiTech Methyl qPCR assay. (C) Cell proliferation assay to measure effects of treatments withtamoxifen or resveratrol alone or in combination on the viability of MDA-MB231 cells; n = 8, *P = 0.0128.

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Fig. S5. STAT3 acetylation levels are highly elevated in human melanoma with ERα silencing. (A) Western blot analysis of ERα in the human melanoma cellsafter treatment with either control or resveratrol. (B) IHC analysis of acetylated STAT3 levels in human melanoma tissues harboring either ERα(+) (un-methylated ERα gene) or ERα(−) (methylated ERα gene); data shown are representative images of tissue sections prepared from 11 ERα(+) and 9 ERα(−)melanoma tumors. (Scale bars, 100 μm.)

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