FPN1 expression is regulated by hepcidin in transfected erythroblast cell lines in a dose- and time-dependent manner as indicated by FACS analysis. (A) Validation of the use of FACS analysis to measure iron-dependent TfR1 expression. K562 cells were treated with 100μM FeNTA or 100μM DFO for 18 hours, and cells were then labeled by APC-labeled TfR1 Abs and checked by FACS analysis. (B) Expression of FPN1 increases TfR1 expression in K562 cells. K562 cells were transiently transfected with pEGFP-N1-FPN1 to express the fusion protein FPN1GFP for 24 hours, and the expression of TfR1 was then measured by FACS. Expression of FPN1 in K562 (C) and MEL (D) cells was down-regulated by hepcidin. Cells were transiently transfected with pEGFP-N1 or pEGFP-N1-FPN1 and incubated with 1 μg/mL hepcidin for 24 hours, and the GFP-positive cells were then measured by FACS. (E) Time-dependent regulation of FPN1 by hepcidin. K562 cells were transfected with pEGFP-N1-FPN1 or pEGFP-N1 vectors for 12 hours, and then incubated with or without 1 μg/mL hepcidin. The GFP-positive cells were measured by FACS at the indicated time points. (F) Dose-dependent regulation of FPN1 by hepcidin. K562 cells transfected with pEGFP-N1-FPN1 or pEGFP-N1 vectors were incubated with hepcidin of indicated concentrations for 24 hours. The GFP-positive cells were measured by FACS. (G) Hepcidin treatment reverses the FPN1-induced increase of TfR1 in K562 cells. K562 cells were transiently transfected with pIRES2EGFP-FPN1 and incubated with or without 1 μg/mL hepcidin for 24 hours. The expression levels of TfR1 were measured by FACS analysis in GFP-positive cells. All of the experiments (A-G) were independently repeated at least twice with triplicate samples assessed at each time point. a.u. indicates arbitrary unit. **P < .01, ***P < .001.