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

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

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

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

High-dose streptozotocin for three days in ZnT-3−/− knockout mice and control mice.

<p>Following a series of low-dose exposures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005684#pone-0005684-g008" target="_blank">Fig. 8</a>) ZnT-3−/− knock-out mice and control mice were treated with 200 mg/kg/day. A) Non-fasting morning blood glucose levels. Salt indicates sham saline injections. Data are mean and SEM (*p<0.01). B) Intraperitoneal glucose tolerance test in ZnT-3−/− knockout mice and control mice after high-dose streptozotozin. Blood glucose concentrations were measured before and 15, 30, 60 and 120 minutes after the glucose injection. Significantly higher AUC_0–120 in knock-out vs WT mice (p<0.01). N = 5 for ZnT-3−/− and n = 4 for wild type.</p

Insulin expression, content and secretion after 24 hours of 100 µM DEDTC treatment.

<p>INS-1E cells were treated with DEDTC at 5 mM glucose. A) Insulin gene expression normalized to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Insulin secretion related to insulin content in INS-1E cells. Data are mean and SEM (*p<0.01). N = 3.</p