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1 Irf7 Fold changes 3 1 Irf1 fold changes 3 1 8h h 8h 8h h 8h p-p6 p-p6 t-p6 p-irf3 β-actin p-irf3 t-irf3 β-actin TKO TKO STKO (E) (F) TKO TKO % of p6 nuclear translocation % % 1% 1% % % p6 TKO % of IRF3 nuclear translocation 1% 8% 6% % % % IRF3 TKO Supplementary Figure 1. qpcr analysis of Irf7 (A) and Irf1 in and MEFs treated with ( µg ml -1 ) or as control for 8 hours. Data are mean of at least three independent experiments. Error bars indicate s.d. ; p<., ; p<.1, Student s t-test. Immunoblot analysis of p6 and IRF3 in and TKO MEFs treated with same as (A) at designated time points; and, TKO, STKO and MEFs treated with same as (A) for hrs. Anti-β-actin was used as loading control; UT: Untreated. Immunofluorescence microscopy analysis of, and TKO MEFs treated with same as (A) for 8 hours using p6 (E) or IRF3 (F) antibody. Images shown are at original magnification (3X) and are representative of at least independent experiments; bar size, µm. Cells from random fields of microscope vision were counted and percentages of nuclear translocation were calculated. Data are mean value of at least three counted fields. Error bars indicate s.d. ; p<.1, Student s t-test.

2 3 CCL mrna Mock Cisplatin Mock Etoposide Cisplatin Etoposide CXCL1 mrna 3 1 NHEKs Ifnβ fold changes 1 1 CTRL NHEKs STING β-actin -h -8h-7h si-c si-sting (E) IFNβ Fold changes (G) IFNβ (pg/ml) sins NHEKs si-sting Mock Cisplatin CTRL cisplatin (F) IFNβ (pg/ml) IFNβ Fold changes (H) sins NHEKs CTRL etoposide si-sting Mock Camptothecin. sicont sicgas. sicont sicgas Supplementary Figure. (A) qpcr analysis of CCL in, TKO, STKO and MEFs treated with either Cisplatin (1 μm) or Etoposide ( μm) for 8 hours. Fluorescence microscopy analysis of PicoGreen staining in MEFs treated same as (A). Images are shown at original magnification, 16X; bar size, 1µm. Normal Human Epidermal Keratinocytes (NHEKs) were treated with µg ml -1 of for hours, 8 hours, or 7 hours followed by qpcr analysis of CXCL1. NHEKs were transfected with STING or control sirna for 8 hours followed by treatment for 8 hours, and then subjected to IFNβ qpcr. Immunoblot analysis of STING expression after sirna treatment was performed using STING antibody and β-actin as loading control (inlet image). NHEKs were sirna treated similarly as in and then treated with Cisplatin at 1µM (E) or Etoposide at µm (F) for 8 hours, followed by qpcr analysis of IFNβ. TKO MEFs were transfected with cgas or control sirna for 8 hours, and then treated with Cisplatin at 1 µm (G) or Camptothecin at nm (H) for 8 hours, followed by IFNβ ELISA analysis. Data are representative of at least two independent experiments. Error bars indicate s.d. ; p<., ; p<.1, Student s t-test.

3 Cell viability (%) hr hr 8 hr 7 hr Cell viability (%) hr hr 8 hr 7 hr AnnexinV positive cells (%) 1 1 TKO STKO TKO STKO Pixels TKO STKO (E) TKO STKO (F) Pixcels TKO STKO (G) TKO STKO (H) nm Gold particles 1nm Gold particles Supplementary Figure 3. (A) Quantitation of cell viability by trypan blue staining of and MEFs treated with µg ml -1 of for hours, 8 hours, and 7 hours. Flow cytometry analysis of, TKO, STKO, and MEFs treated same as (A) for 8 hours and stained with Annexin V. Fluorescence microscopy analysis of PicoGreen staining anti-histone H3 staining (E) in, TKO, STKO and MEFs treated with µg ml -1 of or as control for 8 hours. Images are shown at original magnification, 16X; bar size, 1 µm. Ratio of Cytoplasm to Nucleus PicoGreen pixels and anti-histone H3 pixels (F) was quantitated. (G) Electron microscopy analysis of Histone H3 (6nm Gold particles) and dsdna gold staining (1nm Gold particles) in, TKO, STKO, and MEFs treated same as. Images were shown at original magnification, 1X; bar size, 1nm. (H) Quantitation of gold particles from cytoplasm of three independent cells in each group from (G). Data is representative of at least two independent experiments. Error bars indicate s.d. ; p<., ; p<.1, ; p<.1, Student s t-test.

4 Untreated Skin with no pathological changes Skin with no significant pathological changes Skin with no evidence of tumor or inflammation Exophytic, squamous cell carcinoma, Minimal inflammation, epidermis/dermis Squamous cell carcinoma with keratinization, Mild inflammation, epidermis/dermis Atypical squamous cells without penetration into dermis; Focal area of abscess and ulcer with inflmmation in subcutis,, Inflmmation everylayer, diffuse moderate Skin with squamous cell carcinoma, Mild inflammation, epidermis/dermis Squamous cell carcinoma, Mild inflammation, epidermis/dermis Squamous cell carcinoma, Minimal inflammation, epidermis/dermis Squamous cell carcinoma, exophytic, Inflmmation everylayer, diffuse moderate Squamous cell carcinoma, Mild inflammation, epidermis/dermis Skin with no evidence of tumor, Focal dermal inflammation Skin with no evidence of tumor. Mild dermal inflammation Skin with mild dermal fibrosis. No tumor or inflammation identified Skin with no evidence of tumor. Mild dermal inflammation and fibrosis Skin with hyperkeratosis and dermal fibrosis. No evidence of tumor CD11c positive cells CD68 positive cells Il6 fold induction (E) (F) CCl fold induction Supplementary Figure. (A) Histopathology report of skin/tumor biopsies of treated and mice from Fig. 3. Immunohistochemistry for CD11C and quantitation of CD11c positive cells from same mice in Fig. 3d. Quantitation of CD68 immunohistochemistry analysis in Fig. 3d. Images are shown at original magnification, X; bar size, 1µm. Quantitations were averaged from at least 3 individual mice. Error bars indicate s.d. ; p<., Student s t-test. qpcr analysis of Il6 (E) and Ccl (F) of RNA from skin/tumors same as in Fig. 3e. Data is the mean of three independent mice. Error bars indicate s.d.

5 % Mice free of tumor 1% 1% 8% 6% % % % TKO_ STKO _ Number of tumors TKO_ STKO _ Weeks on treatment Weeks on treatment Group 1 Group logrank p-value significance _ TKO_.71 ns.38 _ STKO_.39 TKO STKO Supplementary Figure. (A) Percentage of skin-tumor free mice and number of skin tumors from, TKO, STKO, and mice treated with 1 μg of on the shaved dorsal weekly for 18 weeks. Results are shown in Kaplan Meier curve. P-value was calculated from log-rank test. Representative photographs of skin tumors and H&E staining of the TKO and STKO mice following treatment with or. The images were shown at original magnification, 1X; bar size, µm.

6 CD7 positive cells Absorbance (nm) mau mau min mau 3 3 Metabolites 1 Metabolites min 1 3 min -h --h --h Supplementary Figure 6. (A) Immunohistochemistry staining of CD7 (Langerin) in skin from and mice treated with or. All images were shown at original magnification, X; bar size, 1 µm. Arrows indicated CD7 positive cells. Quantitation of CD7 positive cells. Data are mean value from at least 3 individual mice. Error bars indicate s.d. ; p<., ; Student s t-test. HPLC analysis of the supernantant from and MEF treated with 1 µg ml -1 of for hours.

7 Untreated thymocytes with dsdna-fitc treated thymocytes with dsdna-fitc 7 6 Cell viability (%) 3 1 No treated treated thymocytes thymocytes CXCL1 Fold changes Untreated thymocytes with dsdna treated thymocytes with dsdna Cxcl1 mrna Supplementary Figure 7. Mouse thymocytes, previously transfected with dsdna-fitc and treated with µg ml -1 of for 8 hours, were added to Bone Marrow Derived Macrophages (BMDMs) isolated from and mice. Macrophage engulfment of thymocytes was then analyzed by fluorescence microscopy 1 hour later (A) and thymocytes viability was estimated by trypan blue staining. CXCL1 expression level in macrophage was analyzed by qpcr 6 hours later. qpcr analysis of Cxcl1 from BMDMs treated with µg ml -1 of for 8 hours. Images are shown at original magnification, 16X; bar size, 1µm. Data are representative of at least two independent experiments. Error bars indicate s.d. ; p<., ; p<.1 Student s t-test.

8 DNA damage Nucleus DNA damage repair Release of DNA into cytosol Cytoplasm ER STING 1 Intrinsic STING-dependent Inflammatory response Cytokines 3 Trex1 -/- STING hyperactivation Apoptosis/Necrosis Immune cell attraction Phagocytosis Extrinsic STING-dependent Inflammatory response Chronic stimulation Inflammation Tumorigenesis Supplementary Figure 8. A schematic illustration of STING-dependent inflammation-driven tumorigenesis by DNA damage response.

9 IFNβ (pg/ml) mock polyi:c dsdna9 STING+/+ STING-/- Myd88-/- IFNβ Fold induction mock polyi:c dsdna9 STING+/+ STING-/- Myd88-/- TNF alpha Fold Induction mock polyi:c dsdna9 CXCL1 Fold induction mock polyi:c dsdna9 STING+/+ STING-/- Myd88-/- STING+/+ STING-/- Myd88-/- (E) (F).3 Myd88 Fold induction MyD88 STING β-actin STING -/- MyD88 +/- MyD88 -/- MyD88+/+ MyD88-/- Supplementary Figure 9. STING +/+, STING -/- and Myd88 -/- MEFs were either mock transfected or transfected with 3 µg/ml of polyi:c or dsdna9. IFNβ expression was then analyzed by ELISA (A), and qpcr analysis was conducted for the gene expression of IFNβ, TNFα and CXCL1. Myd88 -/- genotype was confirmed by loss of Myd88 expression using qpcr (E) and immunoblot analysis (F).

10 Figure 1.a Figure.e Histon H3 STING-HA Β-actin Supplementary Figure 1 Β-actin p-irf3 p-p6 Supplementary Figure 1 p-p6 t-p6 Β-actin p-irf3 t-irf3 Supplementary Figure Supplementary Figure 9(F) Si-STING Myd88 STING STING Β-actin Β-actin Supplementary Figure 1. A list of original immunoblots.

11 Supplementary Table 1. Gene expression fold changes of illumina array shown in Figure 1b. Genes TKO STKO CCL IL1RL ICAM ATF CXCL ADRB PTPN CXCL MDM MDM CCND GZMM CDKN1A CDKN1A PTGES CD MAPK CCND MAPK CCND IFI IRF IL PTGS FAS CDKN1A DUSP FAS ALOX DUSP MAP3K CD PRF DUSP TAP MGST IRF IL Supplementary Table. The category of, Cisplatin, and Etoposide Category Compounds Abbreviation Chemical carcinogens Polycyclic aromatic hydrocarbon 7,1-Dimethylbenzan[A]anthracene Alkylating agent diaminedichloroplatinum(ii) Cisplatin Chemotherapy drugs '-demethyl-epipodophyllotoxin 9-[,6-O-(R)- Topoisomerase inhibitor ethylidene-beta-d-glucopyranoside Etoposide, VP-16 Supplementary Table 3. (A) Number of tumor/mouse from Fig 3b and Number of tumor/mouse from Fig d. (A) mouse # Total 38 8 mouse # BM> BM> BM> BM> Total Supplementary Tables

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