HFt uptake by erythroid cells in various iron statuses.

<p>(A, B) UT7/EPO and HEL cells were incubated with indicated concentrations of FAC or DFO for 24 h before flow cytometry analysis of CD71 (TFR1) expression (upper panels) and HFt (HFt) uptake (lower panels). MFI values relative to those of controls were calculated for each experiment; the results represent the mean ± standard error of at least three independent experiments. *P <0.05, **P <0.01; n.s., not significant.</p

H-ferritin uptake by primary human hematopoietic cells.

<p>(A) Uptake of AF488-labeled human holo-transferrin (Tf), H-ferritin (HFt), and L-ferritin (LFt) by peripheral blood leukocytes. (B) Uptake of AF488-labeled Tf, HFt, and LFt by normal nucleated human BM cells. In (A) and (B), cells were incubated with AF488-labeled ferritins (11 nM) or Tf (62 nM) for 60 min and analyzed by flow cytometry. The MFI ratio was defined as the MFI of cells treated with fluorescence-labeled ligand divided by the MFI of untreated cells. Filled columns denote the results of competition experiments using a 20-fold excess of unlabeled cognate ligand. Data represent the mean ± standard error of three independent experiments. (C) Representative results of HFt uptake by erythroid cells from MDS patients. CD235+ erythroid cells from Patient 1 expressed close to normal levels of CD71 (TFR1) and efficiently incorporated HFt, whereas cells from Patient 2 expressed relatively low levels of TFR1 and incorporated less HFt as compared to cells from normal subjects. Solid lines represent AF488-labeled ligand uptake and shaded areas represent controls.</p

H-ferritin uptake by K562 cells.

<p>(A) Confocal micrographs of AF488-labeled H-ferritin (HFt) and holo-transferrin (Tf). Cells were incubated with 50 μg/ml (110 nM) AF488-labeled human recombinant HFt or 50 μg/ml (625 nM) holo-Tf for 1 h at 37°C. Images are representative results of three independent experiments. (B) Analysis of HFt binding to K562 cells. Cells were incubated with indicated concentrations of AF488-labeled HFt for 60 min on ice, and HFt binding was analyzed by flow cytometry. (C) Competitive inhibition of HFt uptake by K562 cells. Cells were incubated with 11 nM AF488-labeled ferritin in the presence or absence of a 100-fold excess of unlabeled HFt for 60 min on ice, followed by flow cytometric analysis. (D, E) Dose-dependency of HFt and holo-Tf uptake. Cells were incubated with indicated concentrations of AF488-labeled HFt or holo-Tf for 60 min at 37°C, and incorporation of these ligands was analyzed by flow cytometry. (F, G) Time-course of HFt and holo-Tf uptake by K562 cells. Cells were incubated with 11 nM AF488-labeled HFt or 62 nM holo-Tf for indicated times, and incorporation of these ligands was analyzed by flow cytometry. (H) Competition of AF488-labeled HFt uptake with unlabeled ligand. Cells were incubated with 11 nM AF488-labeled HFt and indicated concentrations of unlabeled HFt. (B–H) Data represent the means ± standard errors of three independent experiments.</p

Effect of H-ferritin on holo-Tf uptake by erythroid cells.

<p>(A, B) Competition assays of the TFR1 ligands holo-transferrin (Tf) and H-ferritin (HFt). UT7/EPO (A) or HEL (B) cells were incubated with AF488-labeled holo-Tf or HFt along with the indicated concentrations of unlabeled ligand, and cellular uptake of AF488-labeled ligand was analyzed. Data represent the mean ± standard error of three independent experiments. *P <0.05, **P <0.01; n.s., not significant.</p

TFR1 expression levels required for H-ferritin uptake.

<p>(A) AF488-labeled H-ferritin (HFt) and holo-transferrin (Tf) uptake was evaluated in CHO-TRVb cells lacking endogenous TFR1 (control) and CHO-TRVb cells expressing either wild-type or mutant human TFR1 (R646H and R646H/647A) or wild-type human TFR2. Shaded areas represent isotype control experiments (CD71 expression) and untreated control experiments (Tf and HFt uptake). (B) AF488-labeled holo-Tf and HFt uptake by CHO-TRVb cells expressing moderate or high levels of human TFR1. Representative histograms are shown (left panels). Uptake of AF488-labeled ligand (solid lines), competition experiments using a 20-fold excess of unlabeled ligand (broken lines), and controls (shaded areas) are shown. Data shown in the right panel represent the mean ± standard error. (C) Relationship between CD71 expression and HFt uptake by CHO-TRVb cells expressing wild-type or mutant forms of human TFR1. Data from two cell lines expressing wild-type TFR1 and two expressing a mutated form of TFR1 (R646H/647A) are shown. Right panels show a schematic representation of two different patterns of ligand uptake.</p

Analysis of H-ferritin and holo-transferrin uptake by hematopoietic cell lines.

<p>(A) Uptake of AF488-labeled human holo-transferrin (Tf), H-ferritin (HFt), and L-ferritin (LFt). Cells were incubated with AF488-labeled ferritins (11 nM) or Tf (62 nM) for 60 min and analyzed by flow cytometry. The MFI ratio was defined as the MFI of cells treated with fluorescence-labeled ligand divided by the MFI of untreated cells. Filled columns denote the results of competition experiments using a 20-fold excess of unlabeled cognate ligand. Graphs for NB4, Reh, and THP–1 cell lines are shown on a larger scale in the in the enclosed areas. Data represent the mean ± standard error of three independent experiments. (B) Relationship between HFt and holo-Tf uptake. Cell lines shown in (A) are shown as blue dots by their MFI ratios for HFt and holo-Tf uptake in a scatter plot (Spearman’s coefficient, ρ = 0.95; P < 0.0001). A red dot in this plot represents the position of human bone marrow erythroblasts shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139915#pone.0139915.g003" target="_blank">Fig 3B</a>.</p