The 30 colon carcinoma cell lines utilized were: Caco-2, Colo201, Colo205, Colo320, Dld-1,

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1 Supplementary Text: Materials and Methods Cell lines The 30 colon carcinoma cell lines utilized were: Caco-2, Colo201, Colo205, Colo320, Dld-1, HCT116, HCT-15, HCT-8, HT29, LoVo, LS174T, RKO, SK-CO-1, SW1116, SW403, SW48, SW480, SW620, SW837, SW948, T84, WiDr, HT29-Cl.16E, HT29-Cl.19A, LIM1215, LIM2405, HCC2998, KM12, RW2982 and RW7213. Source and methods of maintenance of the cell lines have been previously described (1). Measurement of Apoptosis Apoptosis assays were performed as previously described by PI staining and FACS analysis (1). Cells were treated with 1, 5 or 10 mm butyrate (Sigma, St. Louis, MO), 1 μm Trichostatin A (Sigma, St. Louis, MO), 2.5 μm SAHA (BioVision, Mountain View, CA) or 10 mm Valproic Acid (Sigma, St. Louis, MO), for 72 hours. Clonogenic assay Cell lines sensitive and resistant to HDACi were treated with 0, 1, 5 or 10 mm sodium butyrate for 24h. Cells were then trypsinized and reseeded in triplicate in 6-well plates at a density of 500 cells/well. Colony formation was monitored over the following 2 weeks. Colonies were stained with 1% crystal violet for 30 min, washed with dh 2 O, air dried, and scanned using a Perfection 1250 flatbed scanner (Epson America Inc., Long Beach, CA). Each cell line was assayed three times, each time in triplicate. Xenografts Xenograft experiments were performed under an IACUC approved protocol. HCT116, HCT8, LIM2405 and HCT15 cells were trypsinized and resuspended in an equal volume of Matrigel (BD Biosciences, San Jose, CA). 5 x 10 6 cells were then injected into the right flank of SCID mice, and monitored for tumor growth. Four animals per group were used. Following development of

2 palpable tumors, animals were injected IP with either PBS or 2.5mg/kg valproic acid, daily, for 2 weeks. Tumor volume was calculated by measurement of the smallest and longest diameter following sacrifice of the animals (2). qrt-pcr Total RNA (5 µg) from colon cancer cell lines treated with HDACi for 24h were reversetranscribed using anchored oligo dt primers and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Primers for specific target genes were designed using Primer 3 software. cdna (10 ng) from each cell line was amplified with specific primers using the SYBR green Core Reagents Kit and a 7900HT real-time PCR instrument (Applied Biosystems). Abundance of gene expression was determined using the standard curve method and expressed relative to GAPDH expression. Primer sequences are provided in Supplementary Table 1. Western blot Western blot analysis was performed as previously described (3). Antibodies for ATF3, Jun, and syntaxin were obtained from Santa Cruz (Santa Cruz, CA) and used at a dilution of 1:200 overnight. Transient transfection assays, sirna knockdown experiments Colon cancer cells were transiently transfected with the Sp1/Sp3 luciferase reporter construct (4) using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA). TK- Renilla was included to control for transfection efficiency. Transfected cells were treated with HDACi for 24 h and luciferase reporter activity determined using the dual-luciferase reporter assay kit from Promega (Madison, WI). As HDACi treatment typically induces both firefly and TK-Renilla luciferase activity, firefly luciferase activity pre and post HDACi treatment was expressed relative to total cellular protein.

3 For sirna experiments, HCT116 cells were transiently transfected using the ProFection transfection reagent (Promega) with a pool of 4 sirnas obtained from Dharmacon (Lafayette, CO). Cells were harvested 24, 48 or 72h post-transfection. ChIP analysis ChIP analysis of the ATF3, EGR1 and JUN promoters was performed in HCT116 cells as recently described (4). In some experiments ChIP analysis was performed in untreated HCT116 cells, whereas for assessment of promoter occupancy pre and post butyrate treatment, untreated cells (24h) were compared to cells treated with 5 mm butyrate for 24h. In all cases 2 x 10 7 HCT116 cells were cross-linked with 1% formaldehyde (Sigma), harvested into cell lysis buffer, and sonicated with 2 x 5 second pulses. The sonicated chromatin was immunoprecipitated with: 5 μg of rabbit polyclonal Sp1 antibody (Upstate Biotechnology), 5 μg of rabbit polyclonal Sp3 antibody (Upstate Biotechnology), 5 μg of anti-hdac3 antibody (Novus Biologicals, Littleton, CO), 5 μg of a rabbit polyclonal acetylated histone H3 antibody (Upstate Biotechnology), or 5 μg of normal rabbit IgG (Santa Cruz). DNA/protein complexes were isolated with Protein A/G Plus agarose beads (Santa Cruz Biotechnology) for 4h at 4 o C, washed, eluted in 0.1% SDS/0.1M NaHCO 3 Elution Buffer, and cross-links reversed overnight at 65 o C in 0.3M NaCl. Input samples were also incubated in this way. DNA was purified using phenol:chloroform extraction and ethanol precipitation. Quantitative real-time RT-PCR was performed, with DNA content in Input and immunoprecipitation samples measured relative to a standard curve of HCT116 cell genomic DNA. All experimental values were expressed relative to relevant Input DNA content. Primers used are listed in Supplementary Table 2. cdna Microarray experiments Exponentially growing cells were treated with 5 mm butyrate for 24 hours. For each cell line, 2 biological replicates were performed, the mean ratio computed for each gene and used in subsequent analyses. Total RNA was isolated from treated and untreated cells using the RNeasy kit (Qiagen,

4 Valencia, CA). For microarray hybridizations, 50 μg of RNA from butyrate treated cells was labeled with Cy5 dutp, and 50 μg of RNA from untreated cells labeled with Cy3 dutp. Probe preparation, hybridization conditions, and array scanning procedure were as previously described (5, 6). Probes were hybridized to 27,000 feature cdna microarrays generated by the Albert Einstein College of Medicine microarray facility (7). Images were analyzed using Genepix Pro software (Axon Instruments, Union City, CA), and lowess normalized. For each cell line, two independent butyrate treatment experiments were performed, and the mean fold-change in expression relative to control computed for each sequence. To identify genes differentially regulated (induced or repressed) in response to HDACi treatment in sensitive and resistant cell lines, the mean fold-change (butyrate relative to control) in expression across the 5 sensitive and 5 resistant lines was computed for each gene. Differentially expressed genes were then selected based upon their ability to satisfy each of the following 3 criteria. First, genes were required to be induced or repressed on average 2-fold or greater relative to untreated controls in either the sensitive or resistant panel. Second, the mean magnitude of change for a gene in the sensitive cell lines was required to be 1.5 fold or greater than the mean magnitude of change in the resistant lines, or vice versa. Third, genes needed to be significantly differentially regulated between sensitive and resistant cells, with a P value <0.05 (unpaired t test) considered statistically significant. To identify genes not changed in response to butyrate treatment, we first identified all genes for which the median fold induction in response to butyrate treatment was less than 10% (within the range of +1.1 to -1.1), for both the sensitive and resistant panel. From this list we then selected genes that were also not significantly differentially expressed between sensitive and resistant lines (p-value resulting from a t-test between sensitive and resistant is greater than.05) clones that meet the above criteria were identified. From these, a random number generator

5 was used to select 48 genes that were used for comparative analyses with the HDACi-induced gene list. Nimblegen Microarray experiments. For comparison of the gene expression changes induced by different HDACi, HCT116 cells were treated with 5 mm butyrate, 5 mm VPA, 5 μm SAHA or 1 mm TSA for 24 hours. Gene expression differences were determined using 2-color Nimblegen microarrays using 10 mg of total RNA from control and treated cells as starting material. Probe labeling, hybridization and image analysis were performed by Nimblegen per standard protocols. Pearson s correlation coefficients were computed for comparison of gene expression changes in response to various HDACi. Human colon tumor / normal specimens Twelve matched pairs of colon tumor and matched adjacent normal mucosa specimens were obtained from patients undergoing surgical resection of colorectal cancer at Montefiore Medical Center, under an IRB approved protocol and with the informed consent of the patient. Specimens were snap frozen in liquid nitrogen and stored at C until use. Total mrna was extracted and subjected to cdna microarray analysis as described above. Genes differentially expressed between normal and tumor tissues were identified using a paired t-test. Bioinformatics Promoter sequences (-10 kb upstream to +10 kp downstream of the Transcription start site) for the 48 HDACi-induced genes as well as an equal number of control genes not changed by HDACi were obtained from the UCSC genome browser ( Once Promoter sequences were obtained, the region encompassing 1.5 kb upstream of the TSS through 200 bp downstream was systematically searched for the presence of transcription factor binding sites using the MATCH tool (BIOBASE Biological Databases GmbH, Wolfenbüttel, Germany) on the TransFac public site. For GC content analyses, each promoter was subdivided into 1 kb windows beginning 10 kb downstream of the TSS and ending 10 kb upstream of the TSS. The GC content

6 of each 1 kb window was computed using the freak tool in the EMBOSS suite accessed on the Washington State University Bioinformatics server: (8). The mean GC content for each window was computed for the HDACi-induced gene set and compared to the un-induced set using an unpaired t test. References 1. Mariadason JM, Arango D, Shi Q, et al. Gene expression profiling-based prediction of response of colon carcinoma cells to 5-fluorouracil and camptothecin. Cancer Res 2003;6324: Wilson AJ, Byun DS, Nasser S, et al. HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21. Mol Biol Cell Wilson AJ, Byun DS, Popova N, et al. Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. The Journal of biological chemistry 2006;28119: Wilson AJ, Byun DS, Nasser S, et al. HDAC4 promotes growth of colon cancer cells via repression of p21. Molecular biology of the cell 2008;1910: Mariadason JM, Corner GA, Augenlicht LH. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res 2000;6016: Mariadason JM, Arango D, Corner GA, et al. A gene expression profile that defines colon cell maturation in vitro. Cancer Res 2002;6216: Cheung VG, Morley M, Aguilar F, Massimi A, Kucherlapati R, Childs G. Making and reading microarrays. Nat Genet 1999;211 Suppl: Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 2000;166:276-7.