Human cxcl12

HEK293 cells (ATCC CRL-11268) were used for the β-arrestin recruitment assay, as previously described^{52 (link)}. In brief, HEK293 cells were transfected with human β-arrestin-2-ω and human CXCR7-α. The cells were stimulated with human CXCL12 (R&D Systems, Minneapolis, MN, USA) or TC14012 at 37 °C for 90 min and then, Gal-Screen (Thermo Fisher Scientific) was added and the cells were further incubated at 25 °C for 90 min. Luminescence was measured in an ARVO X3 plate reader (PerkinElmer, Waltham, MA, USA). In addition, HEK293 cells were transfected with a rat CXCR7 expression plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). The CXCR7 expression plasmid was synthesized by amplifying a rat Cxcr7 mRNA sequence (RefSeq: NM_053352.1) with a FLAG tag (forward primer: 5′-TTTTGCGGCCGCGCCACCATGGATTACAAGGACGATGACGACAAGGGAGGAGGCTCCGATGTGCATCTGTTTGAC-3′, reverse primer: 5′-TTTTAAGCTTTCACTTGGTGTTCTGCTC-3′) from cDNA prepared from total RNA isolated from neonatal rat heart and inserting it in pShuttle-CMV (#16403, Addgene, Watertown, MA, USA). The cells were cultured for 36 h. After 12 h of starvation, the cells were stimulated with CXCL12.

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.