MEF-AOM MEF-DMH Symbol WT-AOM SKO-Mock SKO-AOM Symbol WT-DMH SKO-Mock SKO-DMH EG Rgs1 Ifit3 Ccl3 Usp18 Cxcl2 Rnf2 EG LOC Rgs1

Transcription

1 MEF-AOM Symol WT-AOM SKO-Mock SKO-AOM EG Ifit Usp Rnf LOC Ifit Usp LOC L23Rik Gm D07Rik C530043K16Rik LOC LOC V1rh P16Rik Chst Fn C19Rik A530082L16Rik S100a Nduf Cd K08Rik Calm AU L23Rik D01Rik Ogfod Rnf Rsad Igf2p Gm LOC Peg Olfr Gm Igtp Myo K21Rik C17Rik Zc3h Eif2c Ensa Ell Efna Chrna Kctd Catn F23Rik MEF-DMH Symol WT-DMH SKO-Mock SKO-DMH Rgs Ccl Cxcl EG Rgs Atf Rgs Gdf Ccl EG Aifm Igtp Sprr2a Ra Scyl Zfp Mcm L21Rik Sec11c Usp Ue2t Cxcl Klhdc Slfn Pdh Serhl LOC Rdh Cyp Frag Josd Nkain G10Rik K20Rik Afg3l Coro Rnf Snx Cyr Gp Ngf LOC Myocd Cdkn1a Bet As Ppm1f D07Rik Tappl Rps6k

2 Supplementary Figure S1. The fold change analysis of activated STING-dependent genes from gene array. Wild type (WT) and Sting deficiency (SKO) MEFs were treated with (a) AOM at 0.14mM and () DMH at 1mM for 8 hours. Total RNA was reverse-transcried using M-MLV Reverse Transcriptase. Gene array analysis was examined y Illumina Sentrix BeadChip Array (Mouse WG6 version 2). Highest variale genes are shown. Values are Quantile normalized and log2 transformed to the median of all samples. Significantly differential expressed genes are computed using the Student s t-test and selected using threshold of P-value Hierarchical Clustering and visualization of selected differentially expressed genes is performed on GeneSpring using Pearson Correlation distance method and linkage was computed using the Ward method. Gene expression profiles were processed and statistical analysis was performed at the Sylvester Comprehensive Cancer Center Bioinformatics Core facility University of Miami.

3 WT MEF Mock DMH/DSS IRF3 p65 FHC Mock AOM DMH IRF3 p65 Supplementary Figure S2. The translocation of IRF3/NF-κB y AOM and DMH. (a) Fluorescence microscopy analysis of IRF3 and p65 staining in WT and SKO MEFs. treated with 3mM of AOM and 0.1% DSS or 3mM of DMH and 0.1% DSS. () Immunofluorescence microscopy analysis of FHC treated with AOM and DMH for 48 hours using p65 or IRF3 antiody. Images are shown at original magnification, 1260X.

4 WT SKO # mouse # polyps P= WT SKO Inflammation # mouse score 1 ND 2 ND ND 2 ND P= Supplementary Figure S3. Numer of polyps and micro-endoscopic procedure from WT and SKO mice treated with. WT (n=7) and SKO (n=7) mice were intravenously injected with AOM at a dose 10 mg/kg on Day 1, followed y 7 d administration of DSS at 5 % in drinking water for four DSS cycles. Normal drinking water was used for control group. (a) Numer of polyps On 91 days, micro endoscopic procedure was performed in a linded fashion for counting numer of polyps. () Inflammation score. Mice were sacrificed at day 121 and colon was resected, flushed with PBS, fixed in formalin for HE staining.

5 STING +/+ p53 +/+ STING -/- p53 +/+ STING +/+ p53 -/- STING -/- p53 -/- pbae Ras Myc Ras/Myc Colony numer pbae Ras Myc Ras/Myc STING+/+p53+/+ STING-/-p53+/+ STING+/+p53-/- STING-/-p53-/- Supplementary Figure S4. STING is not involved in Ras- and Myc-induced colony formation in soft agar. Primary MEF cells lacking STING and/or p53 were transduced with retrovirus encoding human H-Ras 12V or human c-myc. After drug selection with puromycin and hygromycin, the cells were cultured in soft agar. After 14 days, colonies were photographed (a) and colony numers in one well (n = 3) were counted (). Error ars indicate SD.

6 TF Position Binding Sequence TAAAGTAAAGCGTATTGTCAATAAATTTCATTGCCACAAAG IRF GAAACTCTGTCAACACCACCACCACCACCAAGAACAAAAGA GAGGCTACATTCTGTGCAATATTGCAATAGTCTGAATGCAA CGCGTTGAGAAGAGGGAGAAGACTAGAAGGAGACAGCTGCA NF-KB CCGAATTTATGGAAAAGTAAAAGTAAAATTTGAAGCTACTT STAT GAGGGACAGTCCAGGCAGTTCTGTGCGTGTTCACTGTTTAG TCTCCCATCTTAGCCTGGGACTCCCATCTGGGACCACAGAT IRF GATAAGAATACATACGTGACTCAAGTTGAAATAGTAAGTTT Supplementary Figure S5. Transcription factor inding sites in IL18 promoter. IL18 promoter region contains inding sites for multiple innate immune gene transcription factors. Putative transcription factor inding sites in the IL18 gene promoter is listed and higlighted in red.

7 IL22p mrna M IL18-10ng/ml IL18-100ng/ml IL ng/ml 0.0 WT SKO Supplementary Figure S6. IL22p mrna expression y in a dose response manner. qpcr analysis of IL-22BP in WT and SKO BMDCs treated with 10, 100, 1000 ng/ml of recominant mouse IL18 (MBL B002-5) for 24 hours. Total RNA was reverse-transcried using M-MLV Reverse Transcriptase. qpcr was performed using Taqman Gene Expression Assay (IL-18 : Mm , IL-22BP : Mm ). Data is the mean of at least 5 mice. Error ars indicate s.d. *; p<0.05, Student s t-test.