Journal: American Journal of Physiology - Cell Physiology

Article Title: The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells

doi: 10.1152/ajpcell.00116.2016

Figure Lengend Snippet: ZIP14 and ZIP8, but not DMT1, overexpression increases iron uptake by βlox5 cells. A: Western blot analysis of cell lysates from βlox5 cells transfected with pCMV-Sport6-empty vector (EV), DMT1, ZIP14, or ZIP8. Tubulin is shown to indicate lane loading. B: effect of ZIP14, ZIP8, or DMT1 overexpression on the uptake of iron by βlox5 cells. To measure iron uptake, cells were incubated for 1 h in serum-free medium containing 2 μM [59Fe]ferric citrate and 1 mM ascorbate and the cellular uptake of 59Fe was measured by gamma counting. Data represent means ± SE of 3 independent experiments performed in triplicate. Group means were compared by unpaired Student’s t-test. *P < 0.05, statistically significant differences relative to cells transfected with EV (ZIP14 P = 0.02, ZIP8 P = 0.005, and DMT1 P = 0.19).

Article Snippet: During the primary antibody incubation, human sections were triple stained for insulin, glucagon, and either DMT1, ZIP8, or ZIP14 by using guinea pig anti-insulin (1:200; Abcam), mouse anti-glucagon (1:1,000; Abcam), and either rabbit anti-DMT1 (1:1,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-Zip8 (1:250; Peprotech), or rabbit anti-ZIP14 (1:1000; Prestige Antibodies; Sigma-Aldrich) antibodies.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells

doi: 10.1152/ajpcell.00116.2016

Figure Lengend Snippet: When overexpressed in βlox5 cells, DMT1, ZIP14, and ZIP8 localize to the plasma membrane. Western blot analysis of DMT1, ZIP14, ZIP8, Na+-K+-ATPase, and copper chaperone for superoxide dismutase (CCS) in total-cell lysate (TCL) or cell-surface (CS) proteins isolated from βlox5 cells transfected with either empty vector (EV), DMT1 (A), ZIP14 (B), or ZIP8 (C). Plasma membrane proteins were labeled with sulfo-NHS-SS-biotin and affinity purified by using streptavidin-agarose columns before Western blotting. Na+-K+-ATPase and CCS serve as markers for plasma membrane and cytosolic proteins, respectively. Images shown are representative of Western blots from 3 independent experiments.

Article Snippet: During the primary antibody incubation, human sections were triple stained for insulin, glucagon, and either DMT1, ZIP8, or ZIP14 by using guinea pig anti-insulin (1:200; Abcam), mouse anti-glucagon (1:1,000; Abcam), and either rabbit anti-DMT1 (1:1,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-Zip8 (1:250; Peprotech), or rabbit anti-ZIP14 (1:1000; Prestige Antibodies; Sigma-Aldrich) antibodies.

Techniques: Western Blot, Isolation, Transfection, Plasmid Preparation, Labeling, Affinity Purification

Journal: American Journal of Physiology - Cell Physiology

Article Title: The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells

doi: 10.1152/ajpcell.00116.2016

Figure Lengend Snippet: NTBI uptake by βlox5 cells is decreased by siRNA knockdown of endogenous ZIP14 but not ZIP8. A: Western blot analysis of lysates from βlox5 cells transfected with negative control siRNA (siNC) or siRNA targeting either ZIP14 (siZIP14, left) or ZIP8 (siZIP8, right). Long exposures of the same blots are also shown to more clearly show the degree of siRNA knockdown. B: to measure NTBI uptake, cells were incubated for 2 h in serum-free medium containing 2 μM [59Fe]ferric citrate and 1 mM ascorbate and the cellular uptake of 59Fe was measured by gamma counting. Data represent means ± SE of 3 independent experiments performed in triplicate. Group means were compared by unpaired Student’s t-test. *P < 0.05, statistically significant differences relative to cells transfected with siNC (ZIP14 P = 0.03 and ZIP8 P = 0.58).

Article Snippet: During the primary antibody incubation, human sections were triple stained for insulin, glucagon, and either DMT1, ZIP8, or ZIP14 by using guinea pig anti-insulin (1:200; Abcam), mouse anti-glucagon (1:1,000; Abcam), and either rabbit anti-DMT1 (1:1,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-Zip8 (1:250; Peprotech), or rabbit anti-ZIP14 (1:1000; Prestige Antibodies; Sigma-Aldrich) antibodies.

Techniques: Western Blot, Transfection, Negative Control, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells

doi: 10.1152/ajpcell.00116.2016

Figure Lengend Snippet: Human islets express ZIP14 and siRNA knockdown of ZIP14 decreases NTBI uptake by primary human islets. A: quantitative RT-PCR analysis of mRNA copy numbers of ZIP14, ZIP8, and DMT1 in isolated human islets. B: Western blot analysis of cell lysates from isolated human islets transfected with either negative control siRNA (siNC) or siRNA targeting ZIP14 (siZIP14). C: to measure iron uptake, cells were incubated for 2 h in serum-free medium containing 2 μM [59Fe]ferric citrate and 1 mM ascorbate and the cellular uptake of 59Fe was measured by gamma counting. Data represent means ± SE of 3 independent experiments performed in triplicate for iron uptake determination and means ± SE of 4 individual islet donors measured in duplicate for mRNA copy number measurement. Group means were compared by unpaired Student’s t-test. *P = 0.002, statistically significant differences relative to cells transfected with siNC.

Article Snippet: During the primary antibody incubation, human sections were triple stained for insulin, glucagon, and either DMT1, ZIP8, or ZIP14 by using guinea pig anti-insulin (1:200; Abcam), mouse anti-glucagon (1:1,000; Abcam), and either rabbit anti-DMT1 (1:1,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-Zip8 (1:250; Peprotech), or rabbit anti-ZIP14 (1:1000; Prestige Antibodies; Sigma-Aldrich) antibodies.

Techniques: Quantitative RT-PCR, Isolation, Western Blot, Transfection, Negative Control, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells

doi: 10.1152/ajpcell.00116.2016

Figure Lengend Snippet: Immunofluorescence analysis of ZIP14, DMT1, and ZIP8 in human pancreatic islets. Human pancreatic tail sections with islets were stained for either ZIP14 (AI, green), DMT1 (BI, green), or ZIP8 (CI, green) along with insulin (A-CII, red) or insulin and glucagon (A-CIII, red and blue, respectively). D: human placenta section stained for ZIP8 and DAPI nuclear stain (DI, green and blue, respectively). Serial sections were analyzed in parallel with nonimmune IgG replacing the primary antibody for ZIP14, DMT1, or ZIP8 (A-CIV: pancreatic sections; DII: placenta section). Images taken at either ×60 (pancreas ZIP14 and DMT1), ×20 (pancreas ZIP8), or ×40 (placenta ZIP8) original magnification were obtained by using a spinning disk confocal fluorescent microscope system. Images shown are from Network for Pancreatic Organ Donation (nPOD) cases 6001 (ZIP14) and 6104 (DMT1 and ZIP8) and are representative of 3–4 individual cases.

Article Snippet: During the primary antibody incubation, human sections were triple stained for insulin, glucagon, and either DMT1, ZIP8, or ZIP14 by using guinea pig anti-insulin (1:200; Abcam), mouse anti-glucagon (1:1,000; Abcam), and either rabbit anti-DMT1 (1:1,000; Prestige Antibodies; Sigma-Aldrich), rabbit anti-Zip8 (1:250; Peprotech), or rabbit anti-ZIP14 (1:1000; Prestige Antibodies; Sigma-Aldrich) antibodies.

Techniques: Immunofluorescence, Staining, Microscopy