Human ccl2

THP-1 cells were counted, resuspended in chemotaxis buffer [0.1% BSA, 10 mM HEPES, 100 U mL⁻¹ penicillin-streptomycin (Life Technologies), and L-glutamic acid (Sigma–Aldrich) in RPMI medium without phenol red (Life Technologies)] and pre-incubated with the inhibitors DSF or cyanamide (Sigma–Aldrich) before chemotaxis assays. Chemotaxis was measured in a 96-well chemoTX chemotaxis chamber with a polycarbonate filter (5-μm pore size) (Neuro probe). Human CCL2 (R&D Systems) was added to the lower chamber of the plate and the cells were added to the upper chamber. After incubation at 37 °C and 5% CO₂ for 90 min, the filter was removed and the number of migrated cells in the lower chamber was counted using a cell counting kit F (Dojindo). Data are expressed as migration efficiency (percentage of the maximal migration).

All recombinant proteins were purchased from R&D Systems, reconstituted as per the manufacturer's instructions and used at the following concentrations: Human CCL2 (R&D Systems, 279-MC-010, 100 ng/mL), human CHI3L1 (R&D Systems, 2599-CH-050, 100 ng/mL), human CXCL12 (R&D Systems, 350-NS-010, 100 ng/mL), human IGFBP3 (R&D Systems, 675-B3-025, 100 ng/mL), human PTX3 (R&D Systems, 1826-TS-025, 1 μg/mL), and mouse Wnt3a (R&D Systems, 1324-WN-010, 25 ng/mL). Cells were incubated in serum-free media for 6 hours before treatment with recombinant proteins. Cells were then treated for 18 hours with serum-free media containing the appropriate recombinant protein with or without the STAT6 inhibitor AS 1517499 (Axon Medchem, 1992-10MG, 50 nmol/L). For Wnt3a treatments, 72 hours incubation was used, and media changed with fresh treatment at 24 and 48 hours after initial treatment.

Antibodies were purchased commercially as follows: mouse CD31 (clone MEC 13.3; BD Pharmingen, San Diego, CA), mouse F4/80 (clone BM8; eBioscience, San Diego, CA), human CD68 (clone PG‐M1; Dako, Glostrup, Denmark), human CCL2 (R&D Systems), mouse iNOS (#ab15323; abcam, Cambridge, MA), mouse arginase1 (#sc‐20150; Santa Cruz biotechnology, Santa Cruz, CA), and mouse MRC1/CD206 (#ab64693; abcam).