Fig 1.
A. Schematic of the targeting construct of Zip4 showing the region of the Zip4 gene flanked by the loxP sites. The cre recombinase induced recombination at these loxP sites which results in the excision of the portion of the Zip4 gene that contains 7 exons. B. PCR results obtained from ear skin samples.
Table 1.
PCR primer sequences used for mouse genotyping and qPCR.
Fig 2.
Localization of Zip4 in MIN6 cells.
A. Field of view of MIN6 cells transfected with the pCMV6-Zip4GFP plasmid. B. Expression of Zip4 was measured by qPCR performed on MIN6 cells transfected with pCMV6-Zip4GFP plasmid C. Co-localization was estimated with the Pearson correlation coefficient analysis calculated from confocal images obtained from the FITC and Cy3 channel. D. Confocal images of pCMV6-Zip4GFP transfected MIN6 cells after staining of the nucleus with TOPRO-3, plasma membranes with WGA Alexa Fluor 555, mitochondria with MitoTracker Red CMXRos or the endoplasmic reticulum (ER) with immunostaining of KDEL.
Fig 3.
Role of Zip4 in zinc transport in MIN6 cells.
A. Fluorescent images (left panels) and 2 representative traces (right panel) of zinc cytoplasmic levels measured with FluoZin-3AM in MIN6 control and pCMV6-Zip4GFP transfected MIN6 cells. B. Following supplementation of 100 μM Zinc, the rate of zinc increase was calculated by applying a linear curve fit and calculating its slope over the first 400 seconds of the fluorescence increase. C. The overall area under the FluoZin-3AM curve in response to 100 μM Zn2+ was calculated in control MIN6 and pCMV6-Zip4GFP transfected MIN6 cells. D. Granular zinc content was estimated with the low affinity zinc dye zinquin in MIN6 cells transfected with pcDNA-eGFP (control condition) and pCMV6-Zip4GFP. E. Overall zinquin intensity in control MIN6 and pCMV6-Zip4GFP transfected MIN6 cells. *. p˂0.05; ***. p˂0.001.
Fig 4.
Role of Zip4 in insulin secretion in MIN6 cells.
A. Glucose stimulated insulin secretion (GSIS) from MIN6 transfected with control vector pCMV-eGFP and MIN6 cells overexpressing Zip4 (pCMV6-Zip4GFP) in presence (10 mM G) or absence of glucose (0 mM G). B. Total insulin content. C. Representative images (left panels) and average mitochondrial membrane potential traces (right panel) monitored in MIN6 cells with Rhodamine123 in absence of glucose (0 mM G), 10 mM glucose (10 mM G), and 10 mM glucose and 20 mM sodium azide (NaN3). D. mRNA expression of genes indicated in MIN6 cells.*. p˂0.05; ***. p˂0.001.
Fig 5.
Localization of Zip4 in the pancreas.
Bright field images of Zip4 immuno-cytochemistry in mouse pancreatic sections.
Fig 6.
Deletion of Zip4 in Zip4BKO mice.
A. PCR results from skin, liver and islet DNA samples showing the wild type (WT), LoxP/LoxP and recombined Zip4 allele. The Zip4 WT allele (187bp) is present in all the different sample of the control RipCre mouse. In the Zip4BKO, LoxP/LoxP Zip4 allele (227bp) is present in all the tissues. Islet DNA of Zip4BKO displays recombined Zip4 gene (400bp) which shows effectiveness and specificity of the Cre lox recombination system. B. qPCR of Zip4 from control RipCre and Zip4BKO isolated islets. C. qPCR was using the ileum as a positive control. D&E immunohistochemistry experiments were performed on dispersed islet cells to co-stain for insulin and Zip4 (D) and glucagon and Zip4 (E). F. Dithizone staining and its quantification in RipCre and Zip4BKO islets.
Fig 7.
In-vivo characterization of Zip4BKO mice.
A. Average body weights of 8 week old RipCre and Zip4BKO mice. B. Oral glucose tolerance test in RipCre and Zip4BKO mice and the corresponding area under the blood glucose curve (C). D. Plasma insulin secretion measured during oral glucose tolerance test. *. p˂0.05.
Fig 8.
mRNA expression of zinc transporters in islets of Zip4BKO mice.
Zip family and Znt8 transcript expression in Zip4BKO mouse islets normalized to control RipCre mouse islets.