1690 hmb

CXCL12 was synthesized using tBoc solid-phase chemistry (39 (link)). Full-length HMGB1 was produced and stored in buffer containing DTT (40 (link)) at the Institute of Research in Biomedicine protein facility, or purchased (1690-HMB, R&D Systems, Minneapolis, MN). PGE₂ (14010) was purchased from Cayman Chemical (Ann Arbor, MI). Recombinant human IL-6 (6206-IL) and recombinant human IL-1β (201-LB) were purchased (R&D Systems, Minneapolis, MN). TG101348 was from Selleck Chemicals (S2736, Houston, TX), celecoxib (PZ0008), and Auranofin (A6733) were purchased from Sigma-Aldrich (Saint Louis, MO). Mouse anti-human CXCR4 APC-conjugated (555976, BD Biosciences, San Jose, CA), mouse anti-human CXCL12 (MAB350, R&D Systems, Minneapolis, MN), rabbit anti-human HMGB1 (Ab18256, Abcam, Cambridge, UK), mouse anti-HMGB1 (HM-901, HMGBiotech, Milan, Italy), and rabbit anti-human thioredoxin (2429, Cell Signaling technology, Leiden, The Netherlands).

BMM were primed using recombinant murine IFNγ (50 ng/mL) (575,302, BioLegend) for 4 h followed by overnight M1 polarization using LPS (100 ng/mL) (L2630, Sigma-Aldrich), recombinant human HMGB1 (rHMGB1) (500 ng/mL) (1690-HMB, R&D), HFHG (500 μM PA in 0.5% BSA and 6 g/L D-glucose), HFHG + rHMGB1. For M2 polarization, the macrophages were stimulated with recombinant murine IL4 (A21323, Antibodies.com) (20 ng/mL) for 24 hs. The macrophages were also stimulated using H9C2 conditioned media for 24 hs. The conditioned medium was obtained following CHOP overexpression, HFHG stimulation in PAK2-knockdown and overexpressing, and Glycyrrhizin (8 h) treated and HFHG (12 h) stimulated cells.

CXCL12 was chemically synthesized as previously described (35 (link)). Full length HMGB1 and HMGB1-Histidine-tagged were produced and stored in phosphate-buffered saline (PBS; D8537, Sigma Aldrich, Saint Louis, MO, United States) at the Institute of Research in Biomedicine Protein Facility (Bellinzona, Switzerland), or purchased (1690-HMB, R&D Systems, Minneapolis, MN, United States). The chosen sub-optimal concentrations of CXCL12 and the concentration of HMGB1 in each experiment were based on our previous findings (6 (link)).