Extracellular sAPPα or CP increases cellular expression of surface ferroportin.

<p>FPN location was examined in (<b>A</b>) HEK293T and (<b>B - D</b>) primary murine neuronal cultures, preincubated with iron (50 µM, 3 h) followed with CP or sAPPα (1 µM, 30 min). Both cell lines have been previously shown to utilize APP to promote iron efflux, and do not express CP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114174#pone.0114174-Duce1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114174#pone.0114174-DeDomenico1" target="_blank">[14]</a>. Surface proteins on (<b>A</b>) HEK293T cells, and (<b>B</b>) primary neurons, were biotinylated to identify changes to endogenous FPN and APP expression on the cell surface, as well as exogenously attached sAPPα or CP. Surface levels of FPN were significantly increased in the presence of CP or sAPPα, despite total levels of FPN remaining unchanged. The graphs show the distribution of FPN when normalized against the β-actin content of the intracellular+surface fractions, and adjusted for protein load. Similar results for FPN distribution were achieved even without adjusting for β-actin (not shown). (<b>C</b>) Fluorescence-activated cell sorting of non-permeabilized N2a neuroblastoma cultures preincubated with iron (50 µM, 6 h) confirms an increase in surface expression of FPN, quantified in (<b>D</b>), after a 30 min incubation with sAPPα (1 µM) in OptiMEM. (<b>E</b>) Deconvoluted confocal microscopy shows overlap of endogenous APP and FPN at the surface of non-permeabilized primary neurons preincubated with iron (50 µM, 3 h), as well as (<b>F</b>) increased FPN on the neuronal surface following further treatment with CP or sAPPα (1 µM, 30 min). Endogenous surface FPN was below detection limits in neurons that were not treated with FAS (not shown). Data in (<b>A</b>), (<b>B</b>) & (<b>D</b>) are means ± S.E. of 3 experiments, performed in duplicate. * p<0.05, ** p<0.01 and *** p<0.001 analyzed treatment vs control, by two-tailed t tests. (<b>C</b>) is a representative histograms of>10,000 live cells normalized to the control (treated with secondary antibody only) mean signal, set at 10². (<b>E</b>) & (<b>F</b>) are representative images of a neuron from 2 experiments, performed in duplicate. Scale bar = 10 µm.</p

Initial rates of reaction for originally purified sAPPα and CP for all 3 outputs in the triplex assay with the absence or presence of TF.

<p>Initial rates of reaction for originally purified sAPPα and CP for all 3 outputs in the triplex assay with the absence or presence of TF.</p

Apparent APP ferroxidase activity derives from the presence of contaminating polyanions.

<p>Recombinant sAPPα was originally eluted from phenyl Sepharose with a buffer (50 mM Na₂HPO₄, pH = 7.4) that lead to a final assay concentration of HPO₄²⁻ at 0.5 mM. Buffer exchanging the sAPPα preparation with HBS prior to its use in the assay (▴) eliminated the presence of this trace polyanion and subsequently ablated activity in all measurements of the triplex assay (<b>A–C</b>). The original iron oxidation rate of the sAPPα (250 nM) preparation (•) was comparable to Na₂HPO₄ alone (), and reintroduction of Na₂HPO₄ with sAPPα (▪) by further buffer exchange produced similar activity to that observed with the original sAPPα preparation. Thus, contaminating HPO₄²⁻ mimicked ferroxidase activity. Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.</p

Contribution of phosphate to iron oxidation from preparations of sAPPα, CP or BSA.

<p>Within the original triplex assay conditions (HBS, pH = 7.2, 26°C), FeSO₄ (100 µM) alone had minimal capability to produce Fe³⁺ within 10 min. The presence of Na₂HPO₄ (0.5 mM) markedly increased the production of Fe³⁺ over the same period. As previously determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114174#pone-0114174-g002" target="_blank">Fig. 2</a>, sAPPα (250 nM) was unable to facilitate Fe³⁺ production unless in the presence of Na₂HPO₄ (<b>A</b>), similar to BSA (<b>C</b>), whereas CP (250 nM) was unaffected and demonstrated greater formation of Fe³⁺ than Na₂HPO₄ alone (<b>B</b>). Ferric ion production was measured as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114174#pone.0114174-Wong2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114174#pone.0114174-Minotti1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114174#pone.0114174-Wong3" target="_blank">[37]</a>. Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.</p

Apparent ferroxidase activity derived from culture medium.

<p>Media of interest was tested by the triplex assay to measure loss of Fe²⁺ (<b>A</b>), conversion of Fe³⁺ (<b>B</b>) and loading of Fe³⁺ into TF (<b>C</b>). Without apo-TF, a one in five dilution of OptiMEM, neurobasal (NB) and RPMI-1640 (RPMI) media oxidized Fe²⁺ to Fe³⁺ (<b>A & B</b>). The addition of apo-TF (50 µM) promoted Fe²⁺ oxidation by all media (<b>A</b>), and had comparable ability to load Fe³⁺ onto TF (<b>C</b>). Assays were run at 26°C in HBS, pH = 7.2+ FAS (100 µM) ± apo-TF (50 µM). Individual data points were mean ± S.E. of 2 experiments, performed in duplicate.</p

Fasting glucose levels after low-dose streptozotozin in ZnT-3−/− knock-out mice.

<p>ZnT-3−/− knock-out mice and control mice were treated with 50 mg/kg/day for five days. Data are mean and SEM (*p<0.01). N = 15.</p

ZnT-3 protein in INS-1E cells and mouse islets.

<p>A) Western blot of ZnT-3 knockout tissue, and normal background strain tissue using the anti-ZnT-3 polyclonal antibody (20 µg per lane). B) Western blot with ZnT-3 antibody. Lane one shows the protein marker in kDa. Subsequent lanes: Control rat brain (10 µg protein) (lanes 2–4), mock transfected INS1-E cells (50 µg protein) (lanes 5–7), 100 µM DEDTC-treated INS1-E cells (50 µg protein) (lanes 8–10), ZnT-3 siRNA transfected INS1-E cells (50 µg protein) (lanes 11–13). Insert shows the quantification, brain tissue values are original multiplied by 5. C) Light micrograph of INS-1E cells exposed to ZnT-3 antibody. Silver enhanced colloidal gold (10 nm) particles attached to secondary antibodies against the ZnT-3 primary antibody are seen within the cells. There was no background stain and controls were negative (insert). Bar = 20 µm. D) Demonstration of ZnT3 antibody positivity in intact mouse islets (lane 1), compared with INS-1E cells before (lane 2) and after (lane 3) treatment with 100 µM DEDTC and brain tissue (lane 4). Each upload with 20 µg protein.</p

Detection of apoptosis in INS-1E cells after 24 hours of hyperglycamia or zinc depletion.

<p>A–C) Glucose stimulation with 5 mM and 16 mM. A) Bax/Bcl-2 ratio of gene expression. Both genes were normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Detection of intracellular DNA fragments in INS-1E cells (apoptosis) after 16 mM glucose stimulation. Data are mean and SEM. N = 4. C) Detection of DNA fragments in medium from INS-1E cells (necrosis) treated 16 mM glucose. Data are mean and SEM (*p<0.05). N = 4. D–F) Zinc chelation with 100 µM DEDTC. D) Bax/Bcl-2 ratio of gene expression. Both genes were normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. E) Detection of DNA fragments in INS-1E (apoptosis) after 100 µM DEDTC treatment. Data are mean and SEM (*p<0.01) N = 4. F) Detection of DNA fragments in medium from INS-1E cells (necrosis) treated with 100 µM DEDTC. Data are mean and SEM (*p<0.01). N = 4.</p

Zinc content of INS-1E cells.

<p>(A+B) Zn²⁺ autometallography of untreated INS-1E cells. (A) 40×. Bar = 50 µm. (B) 100×. Bar = 20 µm. (C+D) DEDTC treatment for 24 hours removed most autometallographically-detectable Zn²⁺ from the INS-1E cells. (C) 40×. Bar = 50 µm. (D) 100×. Bar = 20 µm.</p

Relative gene expression of ZnT-3 and ZnT-8 after 24 hours of 100 µM DEDTC treatment.

<p>INS-1E cells were treated with DEDTC at 5 mM glucose. A) ZnT-3 gene expression normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.05). N = 6. B) ZnT-8 gene expression normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6.</p