Hepcidin knockdown inhibits ferroportin degradation in vitro. (A) Determination of relative abundance of hepcidin expression in THP-1 cells. Cells were left untreated, as a control, or nucleofected with 3 μg of hepcidin siRNA, or, to exclude unspecific effects, nucleofected with a scrambled siRNA. At 36 hours after nucleofection, the mRNA degradation was monitored by real-time PCR. Data are shown as mean plus or minus SD for relative abundances (*P < .001, THP-1 cells nucleofected with 3 μg of hepcidin siRNA vs THP-1 cells transfected with a scrambled siRNA by Student t test). (B) Western blot of THP-1 cells transfected with EmGFP/FP-1 expression vector (lanes 1–4) or mock transfected (lane 5) as a control. Cells were left untreated (lane 1), cotransfected with hepcidin siRNA (lane 2), treated with LPS (1 μg/mL) for 6 hours (lane 3), or cotransfected with hepcidin siRNA and stimulated with LPS (1 μg/mL) for 6 hours (lane 4). Monocytes were subjected to cell membrane extract preparation and subsequent Western blotting for quantification of EmGFP protein expression. Western blotting for β-actin was used as an internal control. One of 4 representative experiments is shown.