Fig. 3

Hepcidin impact on interaction between holo-Tf and Fpn. HEK 293 cells were used to determine the impact of hepcidin on holo-Tf and Fpn interactions using PLA, reported as integrated density per cell in the field of view per image. The level of disrupted interaction was compared to a 0.25 μM holo-Tf and no hepcidin treatment control (A). Cells were co-incubated with holo-Tf and hepcidin for 10 min. The highest concentrations of hepcidin (500 nM) interrupt the interaction between holo-Tf and Fpn when holo-Tf is present at physiological (0.25 μM) levels (D and G), but not at the higher concentrations of holo-Tf concentrations (25 and 2.5 μM) (B and C) or when hepcidin concentrations are closer to physiological baseline of 25 nM (H–J). n = 3 for all experiments, means of biological replicates ± SEM were evaluated for statistical significance using one- way ANOVA with Tukey’s post-test for significance. *p < 0.05, **p < 0.01