Won Jung, Seung Min An, Whaseon Lee-Kwon, Mario Chiong1, Sergio Lavandero1,2, and Hyug

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1 Supplementry Informtion suppresses IL-1-medited immunomodultion Soo Youn Choi, Hwn Hee Lee, Jun Ho Lee, Byeong Jin Ye, Eun Jin Yoo, Hyun Je Kng, Gyu Won Jung, Seung Min An, Whseon Lee-Kwon, Mrio Chiong1, Sergio Lvndero1,, nd Hyug Moo Kwon School of Life Sciences, Ulsn Ntionl Institute of Science nd Technology, Ulsn, Repulic of Kore, 1Advnced Center for Chronic Disese (ACCDiS) & Center for Moleculr Studies of the Cell, Fcultd Ciencis Quimicsy Frmceutics & Fcultd Medicin, Universidd de Chile, Sntigo, Chile, Deprtment of Internl Medicine (Crdiology Division), University of Texs Southwestern Medicl Center, Dlls, Texs, USA Both uthors contriuted eqully to this work.

2 Supplementry Methods DNA ffinity precipittion ssy - Cells were wshed with PBS nd lysed in mm Tris, ph 7.5, 15 mm NCl, 1% Triton X-1, protese inhiitor, 1 mm sodium orthovndte, nd phosphtse inhiitor cocktil for 3 min t 4 C..5 mg/ml of protein ws incuted with 3 nm iotinylted oligonucleotide proe overnight t 4 C in 7. mm Tris [ph7.5], 16 mm HEPES [ph 7.5], 4% glycerol, 166 mm NCl,.4 mm EDTA,.8 mm MgCl, nd.8% Triton X-1. Protein-DNA complexes were isolted using pre-clered streptvidin-grose ed nd nlyzed y western lotting.

3 Supplementry Figures sirna Scr Scr Hsc7 TNFα inos (fold over -) (fold over -) sirna c Protein (pg/ml) IL-4 Protein (pg/ml) IL-13 sirna Supplementry Figure S1 (relted to Figure 1). enhnces M1 gene expression in mcrophges. RAW64.7 cells were trnsfected with scrmled (Scr) or -trgeted sirna followed y tretment with vehicle () or 1 ng/ml for 18 h () or 6 h (, c). () Immunolotting ws performed for nd Hsc7. () Quntittive RT-PCR ws performed for for TNFα nd inos. (c) IL-4 nd IL-13 ws quntified in cell culture medi using ELISA. Men + SD, n = 3. P <.5 compred to corresponding scrmled sirna.

4 Arg-1 CD6 (fold over - IL1(-)-) (fold over - IL1(-)-) sirna IL-1 IL-1 IL-1 IL-1 TNFα (fold over - IL1(-)-) sirna. IL-1 IL-1 Supplementry Figure S (relted to Figure 4). IL-1 enhnces the effects of knockdown on expression of M1 nd M genes. RAW64.7 cells were trnsfected with scrmled (Scr) or -trgeted sirna followed y tretment for 6 h with vehicle () or 1 ng/ml in the presence or sence of murine IL-1 (1 ng/ml). for Arg-1, CD6 nd TNFα were mesured using quntittive RT-PCR. Men + SD, n = 3. P <.1.

5 15 IFNγ Protein (pg/ml) 1 5 Time (min) sirna p-erk ERK p-p38 p38 p-akt AKT Hsc7 Supplementry Figure S3 (relted to Figure 5). knockdown does not ffect IFN-γ secretion nd ctivtion of ERK nd p38 MAPK. RAW64.7 cells were trnsfected with scrmled (Scr) or -trgeted (Ton) sirna followed y tretment with vehicle () or for 6 h () or up to 6 min s indicted (). () IFN-γ in cell culture medi ws quntified y ELISA. Open rs Scr sirna; solid rs sirna. () Totl protein extrcts were sujected to immunolotting for phosphor-erk (p-erk), ERK, phosphor-p38 (p-p38), p38, phosphor-akt (p-akt), AKT,, nd Hsc7. Dt shown re representtive result from three independent experiments.

6 sirna Scr Sp1 Scr DAPA sirna Scr Scr Sp1 Input sirna Scr Scr Sp1 Reltive density Supplementry Figure S4 (relted to Figure 5). knockdown does not ffect the expression nd DNA-inding of Sp1. RAW64.7 cells were trnsfected with scrmled (Scr) or -trgeted sirna followed y tretment with vehicle () or for 1 h. () Totl protein extrcts were sujected to immunolotting for Sp1 nd. () Binding of Sp1 on 5 -iotinylted wild-type Sp1 inding motif ws performed y DNA ffinity precipittion ssy (DAPA) using cell lystes. DAPA smples nd cell lystes (input) were immunolotted for Sp1 nd s indicted. Dt shown re representtive result from three independent experiments.

7 Supplementry Tle Supplementry Tle S1. Gene Forwrd primer Reverse primer Mouse 5 -AAGCAGCCACCACCAAACATGA-3 5 -AAATTGCATGGGCTGCTGCT-3 Mouse IL-1 5 -ACCTGGTAGAAGTGATGCCCCAGGCA-3 5 -CTATGCAGTTGATGAAGATGTCAAA-3 Mouse Arginse-1 5 -CTCCAAGCCAAAGTCCTTAGAG-3 5 -AGGAGCTGTCATTAGGGACATC-3 Mouse CD6 5 -TCTTTTACGAGAAGTTGGGGTCAG-3 5 -ATCATTCCGTTCACCAGAGGG-3 Mouse TNFα 5 -TGGGACAGTGACCTGGACTGT-3 5 -TTCGGAAAGCCCATTTGAGT-3 Mouse inos 5 -GCTCATGCGGCCTCCTTT-3 5 -CCTGTACGGGCATTGCT-3 Mouse IL-4Rα 5 -TGACCTACAAGGAACCCAGGC-3 5 -GAACAGGCAAAACAACGGGAT-3 Mouse SOCS3 5 -AGCTCCAAAAGCGAGTACCA-3 5 -TGACGCTCAACGTGAAGAAG-3 Mouse Bcl-3 5 -GACCTGGAGGTTCGCAATTA-3 5 -CACCATGTTCAGGCTGTTGT-3 Mouse BGT-1 5 -TTCGTGGCTGGGTTTGTTGT-3 5 -ACAGCTGGGACAAAGGCATCAT-3 Mouse SMIT 5 -ATTGCTCATGCCAAAGGCTCCA-3 5 -ACTTGCATGCAGTGTTCTGGGT-3 Mouse Cyclophilin A 5 -CTGCTGTCTTTGGAACTTTGTCTG-3 5 -CAGCCATGGTCAACCCCACCG-3 Humn 5 -AAGCAGCCACCACCAAACATGA-3 5 -AAATTGCATGGGCTGCTGCT-3 Humn IL-1 5 -TTTCCCTGACCTCCCTCTAA-3 5 CGAGACACTGGAAGGTGAATTA-3 Humn Arginse-1 5 -CAGGGCTACTCTCAGGATTAGA-3 5 -CCGAAACAAGCCAAGGTTATTG-3 Humn CD6 5 -GGACGTGGCTGTGGATAAAT-3 5 -ACCCAGAAGACGCATGTAAAG-3 Humn SOCS3 5 -TGGAATGTGTTGGAGGGAAG-3 5 -GGAAGCTGAGGAATTGAAGGA-3 Humn Bcl-3 5 -GGTCAACCCTTCTGGAAACT-3 5 -TGTATGGGTAGGTGCATGTG-3 Humn Cyclophilin A 5 -TTCATCTGCACTGCCAAGAC-3 5 -TCGAGTTGTCCACAGTCAGC-3