HBCs seeded into 6-well culture dishes were incubated with either vehicle control or recombinant human leptin (r-leptin, #300-27-IMG, PeproTech Inc., East Windsor, NJ, USA) at 10, 100, or 1000 ng/mL concentrations in serum-free DMEM/F12 medium supplemented with 1% ITS+ premix (Corning Inc., Corning, NY, USA) for 6 h for mRNA or 24 h for protein analysis. To rule out the potential endotoxin contamination of r-leptin on IL-6 levels, cells were pre-incubated with 1000 ng/mL lipopolysaccharides (LPS) antagonist (LPS-RS; Invivogen, San Diego, CA, USA) for 1 h, followed by 1 ng/mL LPS (L2880, Sigma-Aldrich, Burlington, MA, USA) or 1000 ng/mL r-leptin incubations for 6 h.
To identify relevant signaling pathways involved in leptin-mediated IL-6 expression, HBCs were pre-treated with either control or 1 µM STAT3 inhibitor III, or 50 µM STAT5 inhibitor, 10 µM NF-κB activation inhibitor III, or 20 µM PD98059 as an ERK1/2 inhibitor for 1 h, then treated with either control or 100, or 250, or 1000 ng/mL r-leptin for 6 h for qPCR analysis. All inhibitors were purchased from Millipore (San Diego, CA, USA).
To identify relevant signaling pathways involved in leptin-mediated IL-6 expression, HBCs were pre-treated with either control or 1 µM STAT3 inhibitor III, or 50 µM STAT5 inhibitor, 10 µM NF-κB activation inhibitor III, or 20 µM PD98059 as an ERK1/2 inhibitor for 1 h, then treated with either control or 100, or 250, or 1000 ng/mL r-leptin for 6 h for qPCR analysis. All inhibitors were purchased from Millipore (San Diego, CA, USA).