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Primers used in RT-qPCR, ChIP and Bisulphite-Sequencing. Quantitative real-time RT-PCR primers Supplementary Table S1 gene Forward primer sequence Reverse primer sequence Product TRAIL CAACTCCGTCAGCTCGTTAGAAAG CGGCCCAGAGCCTTTTCATTC 200 IRF1 GCAGGCCCTGACTCCAGCAC TGCCACTCCGACTGCTCCAA 213 STAT1 CTGCTCCCTCTCTGGAATGATGGGTGCATC GAAGTCAGGTTCGCCTCCGTTCTGGGAC 180 DR5 TCCACCTGGACACCATATCTCAGAA TCCACTTCACCTGAATCACACCTG 132 DR4 CTGCTGCAGGTCGTACCTAGCTC GATCCTGGTGGACACAACTCTCC 110 APAF1 GCAGCCAGCTTCAGGATCTAC CAAAGTTCCTTGTGCATCTTGG 154 BID TGGACTGTGAGGTCAACAACG AGTCTGCAGCTCATCGTAGCC 176 CASPASE 8 TTTCTGCCTACAGGGTCATGCTC GTTCATGTCATCATCCAGTTTGC 123 XAF1 GCTCGGGAAAGGGGAAAGAATTT TCTGAGTCTGGACAACATTTACCC 120 DcR1 TCCTGCTGCCAGTCCTAGCTTAC TGAGATCCTGCTGGACACTCCTC 126 DcR2 GCCGGTCCGGGTTGACTC TGAGATCCTGCTGGACACTCCT 117 BCL2 TGGGATGCCTTTGTGGAACTGTA ATATTTGTTTGGGGCAGGCATGT 176 ciap1 TCAGCTAGTCTGGGATCCACCTC AGGGTTTGGAGAAAGGCTGGAGT 120 ciap2 TTCCGGGTACAGAAAACAGTGGA TGGTAGGAACTTCTCATCAAGGCAGA 115 XIAP GCAAGAGCTGGATTTTATGCTTTAGG CAGATATTTGCACCCTGGATACCAT 138 cmyc CCCGGAGTTGGAAAACAATGAAA GTCGTTTCCGCAACAAGTCCTCT 128 EGFR GGTGGCATTTAGGGGTGACTC CTGACCATGTTGCTTGGTCCT 187 ERBB2 GACTGCCTGTCCCTACAACTACC CCCAGACCATAGCACACTCG 150 CCND1 CCCTCGGTGTCCTACTTCAAA AGGAAGCGGTCCAGGTAGTTC 149 SHH CCAGAAACTCCGAGCGATTTA ATGGCCAAAGCGTTCAACTT 133 PAX-2 CAAATCAGAACAGGGGAACGA GCCATGTCACGACCAGTCAC 153 cmet GGTTGTGGTTTCTCGATCAGG TTCGTGATCTTCTTCCCAGTGA 144 GAPDH CATGGCACCGTCAAGGCTGA TGGTGGTGCAGGAGGCATTG 295

TRAIL (mouse) TCCAATCTCCAAGGATGGAAAGA GATGTAATACAGGCCCTCCTGCTC 139 DR5 (mouse) TGCTGTGCTACAGGCTGTCTTTG CTGACAGGTACTGGCCTGCTAGA 125 36B4 (mouse) AATCTCCAGAGGCACCATTG CCGATCTGCAGACACACACT 252 ChIP primers gene Position relative to the TSS Forward primer sequence Reverse primer sequence Product TRAIL 1-2107 -> -1845 GCCGACTCTTGTAACTCCTCAAAT TATGATGTACGTGAAAGGCATGAA 163 2-1480 -> - 1292 GATAGAAGGCAAGGGCAGGAAGTG GTTTAGTTATTCCATTCTTGACCCACTG 189 3-803 -> -607 CATCTGATAGTGGGGAGATTTGG TGTTAGGCTGGACAGGTAGGAAG 197 4-144 -> +51 AGCTTCTTTCAGTTTCCCTCCTTT TCACTGAAGCCCTTCCTTCTCTAT 195 5 +629 -> + 793 CAGGTTCTTTGGTGCCCATT TCGCAGGAATTCTCATGTCC 165 6 +5293 -> +5495 CTACCTTCTGCCCCCAATGC GAACTTGGCCAATGCTGGTG 203 MYO -1543 -> -1374 TGGCACATAAAGTTCTTCCCAGTA TAGGAGTTTGTTCAGAGGTGATGG 170 MYOD1 + 580 -> +654 CCGCCTGAGCAAAGTAAATGA GGCAACCGCTGGTTTGG 75 IRF1-208 -> -44 CGGAGCAGCCGCCCTGTACTTCCCCT TCGCC GCGCCACCGAGCAATCCAAACACTTAGCG 165

Primers used for PCR amplification before bisulphite sequencing Sequence T A ( C) PCR product Region amplified TRAIL Intron1/Exon1 Region A Forward 5 -GATTATGGTTATGATGGAGGTTTAG-3 56 240 +91 +330 Reverse 5 -AAAACAAAAAAATCTTCAAAAAAAA-3 TRAIL CpG island 3 Region B Forward 5 -AATATAGGGGAGAAGATTAAAGAAA-3 56 278-2052 -1773 Reverse 5 -CCTCCTAAAATACTAAAATTACAAA-3 TRAIL CpG island 5 Region C Forward 5 -TGTAATTTTAGTATTTTAGGAGGT-3 58 144-2171 -2028 Reverse 5 -TAAAACTACAAACACCCACCAC-3 Sequences of the primers used for amplification of bisulphite-modified DNA are listed with the annealing temperature (T A ) used. In addition, the length of the PCR product and the location of the region amplified relative to the transcriptional start site (TSS) (+1) are indicated.

Supplementary Figure Legends Lund et al. Transformation-dependent Silencing of Tumor-selective Apoptogenic TRAIL by DNA Hypermethylation is Antagonized by Decitabine Figure S1. Transcription-factor binding and histone-modifications at the TRAIL promoter of HA1E and HA1ER. A, Illustration of the TRAIL proximal promoter with the positions of transcription factor binding sites relative to the transcription start site (TSS; +1) indicating the locations of the regions analyzed by qpcr after ChIP. B, ChIP assays on the TRAIL promoter performed with antibodies against STAT1. The fold enrichment is the relative abundance of DNA fragments at the indicated regions over a transcriptional inactive control region (TSS of Myoglobin), n = 2. E-J, ChIP assays on the TRAIL promoter performed with antibodies against E, H3K27me3; F, H3; G, H3K4me1; H, H3K9me1; I, H3K9me3 and J, H3K27me1. The fold enrichment at known target sites is shown to the right hand of the graphs for B, the STAT1 binding at the IRF1 promoter, for C and D, the SP1 and SP3 binding at the DHFR promoter and for E, the H3K27me3 enrichment at the MyoD1 promoter. HA1E and HA1ER cells were either untreated (gray bars) or incubated with IFNγ (10 ng/ml; 18h; black bars). Bars indicate the fold of enrichment that is the relative abundance of DNA fragments at the indicated regions over a transcriptional inactive control region (TSS of Myoglobin), n = 2. Data are means ± SD. Figure S2. TRAIL induction through DNA demethylation of promoter regions by decitabine. A, Time-course of TRAIL mrna expression after treatment with decitabine in HA1E and HA1ER measured by RT-qPCR, n = 2. Data are means ± SEM. B, Bisulphite-Sequencing of the Exon1/Intron1 TRAIL promoter region (right) and the region covering the 5 part of the CpG-island 2kb upstream of the TSS (left). Comparison of per cent methylation for each

individual CpG (numbered in 5 to 3 direction) in untreated HA1ER (gray bars) versus decitabine treated HA1ER (black bars) is shown. C, Representation of the Bisulphite sequencing results displaying all single clones analysed. The individual CpG-sites (circles) are depicted as methylated (black) or unmethylated (white). Figure S3. Synergistic apoptosis induction by combination of TRAIL and decitabine. Light microscopic images of HA1ER treated with TRAIL, decitabine or a combination of both drugs demonstrates synergistic apoptosis induction by the combinatorial treatment; 20x magnification. Figure S4. TRAIL induction and antitumor effect of decitabine in syngeneic mice models. A, A, RT-qPCR analysis for TRAIL- and DR5-induction after treatment with decitabine, n = 3 and corresponding Western blot for TRAIL in C26 mouse colon tumor cells. B, Decitabine reduces tumor growth in a syngeneic BALB/c tumor model. Upper Left: Normalized tumor volume of C26-grafted mice after treatment with decitabine. Upper right: RT-qPCR for TRAIL across tumor samples, n = 3. Bottom: Normalized animal weight profile in in C26- syngeneic tumors. Administration of decitabine (1.5) (black squares) does not cause toxicity as observed by the lack of change in body weight of animals. The fold of mrna induction is normalized to the untreated cells (set to 1). Samples were taken at the day of sacrifice. Data are means ± SEM. *P <0.05, **P <0.01, ***P <0.001. Figure S5. Regulation of proto-oncogenes by decitabine. RT-qPCR analysis for regulation of proto-oncogene mrna levels after treatment with decitabine (10µM) for 96 hours, n = 3. The fold change is indicated relative to the untreated cells (>1 = Up-regulation, 1 = unchanged, < 1 = down-regulation). Data are means ± SEM.

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