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	Figure 1. NA-59Fe(II) transport in Caco-2 cell monolayers is pH-sensitive and concentration-dependent. (A) structure of nicotianamine (NA) and the estimated coordination manner of NA–iron complex. (B) 59Fe radioactivity in the monolayer Caco-2 cells determined after the addition of 100 μM 59Fe(II) or NA-59Fe(II) containing 1 mM NA and 100 μM Fe(II) at pH 6.0, 7.4, or 8.0 (n = 7, ∗∗∗p < 0.001, ∗∗p < 0.01). (C) Caco-2 cells (n = 5) and the culture solution on the basal side (n = 4) were collected and counted 30 min after the addition of 100, 200, or 500 μM NA-59Fe(II) or 59Fe(II) (∗p < 0.05).
	Figure 2. hPAT1 expression in oocytes detected by immunohistochemistry using anti-hPAT1 polyclonal antibody staining. (A) and (C), hPAT1 expression (red) was detected using anti-hPAT1 antibody on the plasma membranes of oocytes (C) injected with hPAT1 cRNA. (B) and (D), negative control (injected with water). (C-D) simultaneous immunostaining (green) with the plasma membrane marker Ca2+ ATPase (PMCA) was performed. The outer surfaces of the oocytes are indicated by blue arrows. DIC, differential interference contrast. Scale bar indicates 100 μm.
	Figure 3. Electrophysiological NA-59Fe(II) transport assays with Xenopus oocytes expressing hPAT1. An oocyte was voltage-clamped at −60 mV during the current recording while the ND96 buffer (pH 6.0) was perfused. (A) iron transport activities of oocytes injected with hPAT1 cRNA or water were measured in 100 μM NA-Fe(II) (1 mM NA:100 μM Fe), 100 μM NA or proline (Pro) (n = 6). (B) as a positive control of NA-Fe(II) transporter activity, ZmYS1 cRNA or water was injected into oocytes, and the currents were measured in the presence of 100 μM NA-Fe(II). (C) currents induced by various concentrations of NA-Fe(II) (0, 10, 20, 50, 100, and 200 μM) (n = 3–6, ∗p < 0.05; ∗∗p < 0.01). (D) inhibitory effects of 1 or 5 mM 5-hydroxytryptophan (5-HTP) against NA-Fe(II)-elicited current were evaluated for 0.5 mM NA-Fe(II) (n = 3–5, ∗∗∗p < 0.001, ∗p < 0.05).
	Figure 4. Iron uptake in hPAT1-knockdown Caco-2 cells. (A) hPAT1 mRNA expression in RNAi-mediated hPAT1-knockdown Caco-2 cells (dotted bars) was measured by qRT-PCR. The control cells (white bars) were transformed with a Neg-miRNA plasmid (n = 3, ∗p < 0.05, ∗∗p < 0.01). (B) western blotting of Caco-2 cells transfected with plasmid only (column c) and hPAT1 RNAi showing hPAT1 and GAPDH levels. The calculated molecular weights of PAT1 and GAPDH are about 53 kDa and 36 kDa respectively. The images were obtained using an Amersham Imager 600. (C) 59Fe isotope contents (as percentage of the control value) detected in hPAT1-knockdown Caco-2 cells, the radioactivity was measured after the addition of 100 μM NA-59Fe(II) (n = 3, ∗p < 0.05). (D) 59Fe isotope contents detected in hPAT1-knockdown Caco-2 cells; radioactivity was measured after the addition of free 59Fe(II) (100 μM) (n = 3).
	Figure 5. Comparison of59Fe and Hb contents after oral administration of Fe and NA-Fe(II).59Fe radioactivity was measured in the blood, liver, kidney, spleen (A), and small intestine at 0.5 h (B), 2 h (C), and 5 h (D) after oral administration of NA-59Fe(II) or 59Fe(II) (n = 7–9, ∗p < 0.05, ∗∗p < 0.01). The vertical axis is 59Fe counts for each organ per gram weight that was expressed as the percentage of total activity of 59Fe administered to mice. (E) hemoglobin (Hb) levels in whole blood of mouse were measured at 0 h, 0.5 h, 2 h, and 5 h after oral administration of NA-59Fe(II) or 59Fe(II) (n = 4, ∗p < 0.05).
	Figure 6. Relative expression levels of PAT1 and DMT1,59Fe and NA contents in various sections of mouse intestine. (A) mouse small intestine was divided into ten sections (sections numbered 1–10) that were defined as the duodenum (section 1), proximal jejunum (sections 2–5), distal jejunum (sections 6–7), and ileum (sections 8–10). (B) mRNA levels of PAT1, (C) DMT1 as determined by qRT-PCR in the duodenum (section. 1), proximal jejunum (sections 2–5), and distal jejunum (sections 6–7). The relative expression levels were calculated by setting the average values obtained for section 1 to 7 (n = 3–4, ∗∗p < 0.01, ∗p < 0.05). Uptake of 59Fe was determined by radioactivity measurements in the upper intestine sections 1 to 7 30 min after oral administration of NA-59Fe(II) (D) or 59Fe(II) (E) (n = 4, ∗p < 0.05). F, amounts of NA obtained from each section of the small intestine. Quantification of NA levels in these extracts was performed by quantitative analysis with LC-MS after FMOC conversion. (n = 4, ∗p < 0.05).

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