Recombinant human resistin

Human resistin transgenic mice (hRetnTg⁺) were generated as previously described by Mitchell Lazar [26] (link). Briefly, the human resistin gene, along with 21,300 bp upstream and 4,248 bp downstream of the human resistin start site, were engineered through a bacterial artificial chromosome. hRetnTg⁻ mice were backcrossed onto WT C57BL/6 mice bred in house to generate mRetn^−/− mice. In the resistin binding assay, CX3CR1^GFPPGRP^dsred mice were used as reporters for monocytes and neutrophils respectively [58] (link). Recombinant human resistin (Peprotech) was used for in vivo intraperitoneal injections (500 ng/mouse at days -1 and/or day -3). In some experiments, mice were also treated intratracheally with 500 ng hResistin. Cells from the peritoneal cavity were recovered at 24, 48, and 72 hours and analyzed by flow cytometry and real-time PCR. Recombinant hResistin was tested for endotoxin contamination using the Pierce Limulus Amebocyte Lysate assay (Thermo Scientific) according to manufacturer's instructions under sterile conditions. For controls, mice were injected i.p. with PBS or the limit of detection concentration of LPS (1.5pg/mL). All animals in the experiment were age-matched (6–10 week old) and gender-matched, and housed five per cage under ambient temperature with 12 hour light/dark cycle.

FLSs were cultured overnight in 96-well plates (2 × 10⁴ cells/well) and then incubated with recombinant human resistin (0, 10, 100, or 1000 ng/ml; PeproTech) at 37 °C for 24 h in RPMI1640 medium containing 1% FBS. Concentrations of CXCL8, CCL2, IL-1β, IL-6, and IL-32 in culture supernatants were assessed using the ELISA kit (R&D Systems), according to the instructions of the manufacturer.

Human recombinant resistin and VEGF-A protein were purchased from PeproTech (Rocky Hill, NJ, USA). VEGF-A antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies for resistin, ERK, JNK, p38 and p65 (all mouse monoclonal IgG antibodies) and for pERK, pJNK, pp38 and pp65 (all rabbit monoclonal IgG antibodies) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SP600125, SB203580, PDTC and TPCK were purchased from Calbiochem (San Diego, CA, USA). ON-TARGETplus small interfering RNAs (siRNAs) of JNK, ERK and p38 were purchased from Dharmacon Research (Lafayette, CO, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Reporter lysis buffer was purchased from Promega (Madison, WI, USA) and human osteosarcoma tissue arrays were purchased from Biomax (Rockville, MD, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).