Relative quantification was performed

using the comparative Ct, or ddCt method.18 The target gene (Hepc1 or ID1) was first normalized to a reference housekeeping gene (β-actin) and then presented as the difference from the control treatment within each experiment. The average HM781-36B cost ddCt value of the triplicate control treatments was zero in each experiment. Cells for whole-cell lysates were plated in 60-mm collagen-coated dishes (BD Biocoat, Franklin Lakes, NJ) and cells for fractionated lysates (nuclear and cytosolic) were plated in 10-cm collagen-coated dishes. After treatment, whole-cell lysates were collected in ice-cold NETT buffer (150 mM NaCl, 10 mM EDTA, 10 mM Tris, 1% Triton X-100) containing HALT protease/phosphatase inhibitor cocktail (ThermoScientific/Pierce, Rockford, IL). Fractioned lysates were separated into nuclear and cytosolic fractions using the NE-PER kit (Pierce) according to the manufacturer’s instructions. Lysates (30 μg of fractionated lysates, 50 μg of whole-cell lysates) were electrophoresed on 4%-20% LongLife iGels (NuSep, Lawrenceville, GA) and transferred to Immobilon-P PVDF membrane (EMD Millipore) for western blotting,

visualization by chemiluminescence, and quantification with the ChemiDoc XRS+ System with Image Lab software (BioRad, Hercules, CA). Twenty-four hours after isolation, serum-starved hepatocytes were pretreated with kinase inhibitors in a 0.5 μL volume of dimethyl sulfoxide (DMSO). Each kinase inhibitor was added to duplicate or triplicate wells and after 1 hour 20 ng/mL HGF was added, then 1 hour later 10 ng/mL BMP6 was added. Depending on the kinase inhibitor, Dabrafenib mouse cultures were incubated for a minimum of 8 hours or overnight

prior to sample collection. WT 6-week-old C57BL/6 mice were mildly iron-depleted by being placed on a diet with less than 4 ppm iron (Harlan Teklad) for 7 days prior to the experiment. On the day of injection, mice received a series of three intraperitoneal injections (spaced 6 hours apart) of 50 μg human EGF (Millipore) or saline. Some mice were injected with 5 mg holotransferrin (Sanquin, Amsterdam, The Netherlands) with the third dose of EGF or saline. Liver samples were collected 24 hours after the first dose of EGF was administered. HGF dose-dependently suppressed hepcidin mRNA in MCE公司 hepatocytes (Fig. 1A). When hepcidin was induced by its physiological stimuli, holotransferrin or BMP, HGF significantly lowered both baseline hepcidin expression and the maximal induction of hepcidin by holotransferrin (Fig. 1B) or by a range of BMP6 concentrations (Fig. 2A). At each concentration of BMP6, HGF addition caused 10- to 20-fold suppression of hepcidin mRNA. In experiments where IL-6 was used as the inducing cytokine, HGF suppression of hepcidin mRNA was overcome by increasing concentrations of IL-6, even though it is a less potent hepcidin inducer than BMP6 (Supporting Fig. S1). Among other growth factors tested, only EGF suppressed hepcidin mRNA similarly to HGF (Fig. 2B). PDGF (Fig.