Supplemental Materials and Methods s3

Supplemental Materials and Methods

Materials

LXA4 and U-75302 (6-(6-(3R-hydroxy-1E,5Z-undecadien-1-yl)-2-pyridinyl)-1,5S-hexanediol) were obtained from Cayman Chemical (Denver, CO). Recombinant human SAA, M-CSF and MCP-1 were from PeproTech (Rocky Hill, NJ). The LL-37 peptide

(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was chemically synthesized by GenScript

Corporation (Piscataway, NJ, USA). lipopolysaccharides (LPS; from Escherichia coli strain 0111:B4),

ANXA1, diphenyleneiodonium (DPI), pertussis toxin (PTX), WRWWWW-NH2 (WRW4), dimethylfumarate (DMF) and Cell Counting Kit-8 (CCK-8) were bought from Sigma (Copenhagen,

Denmark). E5564 (also known as B1287) was synthesized at the Eisai Research Institute (Andover,

MA). Radioiodinated SAA (125I-labeled) which was prepared by SAIC Frederick (NCI-FCRDC,

Frederick, MD). NSC74859 (S3I-201) was purchased from EMD Chemicals, Inc. (Norwood, OH).

Lipofectamine 2000 and Trizol Reagent were from Invitrogen (Carlsbad, CA). Luciferase assay system, galactosidase enzyme assay system, oligo dT primer, moloney murine leukemia virus (M-MLV) reverse transcriptase, the set of dNTPs and RNase inhibitor were all purchased from Promega

(Madison, WI). The NF-kappaB-Luc reporter plasmid (pNF-kappaB-Luc) was purchased from

Stratagene (La Jolla, CA). SYBR Green I PCR Master Mix was the product of Applied Biosystems

(Foster City, CA). The primary antibodies rabbit anti-HIF-1, COX-2, NF-kB p50, -actin were purchased from BD Biosciences (San Jose, CA), rabbit anti-pSTAT3 (Tyr705) and SOCS3, mouse anti- pERK and pp38 were from Cell Signaling Technology, Inc. (Beverly, MA). RNA knockdown experiments

Lentivirus short hairpin RNA vector of formyl-peptide receptor 2 (LVshFPR2) and negative control

(scrambled) sequences were designed, synthesized, and labeled with RFP (GeneChem, Inc., Shanghai, China) and the former were utilized to specifically knockdown FPR2 expression by HepG2 cell line. The targeted exon sequences of FPR2 and scrambled were CACATACTGTACTTTCAAC and TTCTCCGAACGTGTCACGT, respectively. For each of the LVshRNA constructs, the HepG2 cells were transfected with lipofectamine-2000 reagent, after which these cells were utilized for the functional studies 24-48 h later.

siRNA targeting NF-kB p50 was designed according to Tuschl’s method found online http://www.rockefeller.edu/labheads/tuschl/sirna.html. The sequence of p50 and scrambled siRNA were 5’-

TATTAGAGCAACCTAAACATT-3’ and 5’-CCTACGCCACCAATTTCGT-3’, respectively. Scrambled siRNA was used as a non-silencing control siRNA. All siRNAs were resuspended in DEPC-treated water to a final concentration of 30 μM. For each transfection, 3.5 μl of INTERFERin (Polyplus, Strasbourg, France) was mixed with 250 μl of Opti-MEM and incubated for 5 min at room temperature. In a separate tube, 0.5 pmol of siRNA was added to 250 μl of Opti-MEM and the siRNA solution was added to the INTERFERin mixture. The siRNA/INTERFERin mixture was incubated for an additional 20 min at room temperature to allow complex formation. Subsequently, the solution was added to the cells in the 6-well plate, giving an end volume of 1 ml.

After 36 h incubation with the transfection solution, cells were harvested for Western-blot and other experiments.

Supplemental Table 1. Primers used for real time-PCR Gene name Orientation Primer sequence (5' - 3') Species M-CSF forward TAGCCACATGATTGGGAGTG human M-CSF reverse TATCTCTGAAGCGCATGGTG human MCP-1 forward GCTGTGATCT-TCAAGACCATTGTG human MCP-1 reverse TGGAATCCTGAACCCACTTCTG human FPR2 forward AACCCCATGCTTTACGTCTTTGTG human FPR2 reverse ATTGGCAGCCGTGTCATTAGTTG human GAPDH forward ACCAGCCCCAGCAAGAGCACAAG human GAPDH reverse TTCAAGGGGTCTACATGGCAACTG human M-CSF forward CATCCAGGCAGAGACTGACA mouse M-CSF reverse CTTGCTGATCCTCCTTCCAG mouse MCP-1 forward CTTCTGGGCCTGCTGTTCA mouse MCP-1 reverse CTTCTGGGCCTGCTGTTCA mouse IL-10 forward TGAGGCGCTGTCGTCATCGATTTCTCCC mouse IL-10 reverse ACCTGCTCCACTGCCTTGCT mouse IL-12p35 forward CACCCTTGCCCTCCTAAACC mouse IL-12p35 reverse CACCTGGCAGGTCCAGAGA mouse IL-23p19 forward CAGCAGCTCTCTCGGAAT mouse IL-23p19 reverse ACAACCATCTTCACACTGGATACG mouse IL-6 forward GAGGATACCACTCCCAACAGACC mouse IL-6 reverse AAGTGCATCATCGTTGTTCATACA mouse IL-1β forward CAACCAACAAGTGATATTCTCCATG mouse IL-1β reverse GATCCACACTCTCCAGCTGCA mouse TNFα forward CACGTCGTAGCAAACCACCAAGTGGA mouse TNFα reverse TGGGAGTAGACAAGGTACAACCC mouse CCL17 forward TAAGACCTCAGTGGAGTGTTC mouse CCL17 reverse AAATGCCTCAGCGGGAAGC mouse CCL1 forward CCAGACATTCGGCGGTTG mouse CCL1 reverse CAGCAGCAGGCACATCAG mouse CXCL13 forward CTCTCCAGGCCACGGTATT mouse CXCL13 reverse TAACCATTTGGCACGAGGAT mouse CXCL8 forward CTCTTGGCAGCCTTCCTGATT mouse CXCL8 reverse TATGCACTGACATCTAAGTTCTTTAGCA mouse GAPDH forward TGCACCACCAACTGCTTAG mouse GAPDH reverse GGATGCAGGGATGATGTTC mouse Supplemental figure legends

Figure S1. Effects of FPR2 agonists on M-CSF and MCP-1 expressions induced by LPS. After

HepG2 cells were treated with LPS (1 g/ml) in the absence and presence of LXA4 (100 nM), SAA

(100 nM), ANXA1 (100 nM) or LL-37 (50 g/ml) for 24 h, the mRNA expression (A) and protein secretion (B) of M-CSF and MCP-1 were detected with real-time PCR and ELISA respectively.

*P<0.05 and **P<0.01 vs. control; #P<0.05 vs. LPS group.

Figure S2. Effects of ANXA1 and LL-37 on the mRNA expressions of tumor infiltrating macrophage phenotypes. Mice were treated as decribed in Materirals and Methods. The mRNA expressions of macrophage phenotypes were detected with real-time PCR. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. H22 group.

Figure S3. Macrophages densities in tumor xenografts. Tumors were derived from Balb/c mice transplanted with H22 tumor. ANXA1 or LL-37 was injected i.v., WRW4 was given i.p. as indicated in

Materials and Methods. The tissues were cut into fragments. After digested with collagenase, the cells were then passed through a 70 μm mesh to provide a single cell suspension and resuspended in calcium and magnesium free HBSS. Cells were stained with anti-CD68-PE and analyzed with flow cytometry.