Role of Zip4 in insulin secretion in MIN6 cells.

<p>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.</p

Generation of Zip4BKO mice.

<p>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.</p

In-vivo characterization of Zip4BKO mice.

<p>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.</p

Localization of Zip4 in the pancreas.

<p>Bright field images of Zip4 immuno-cytochemistry in mouse pancreatic sections.</p

Deletion of Zip4 in Zip4BKO mice.

<p>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.</p

mRNA expression of zinc transporters in islets of Zip4BKO mice.

<p>Zip family and Znt8 transcript expression in Zip4BKO mouse islets normalized to control RipCre mouse islets.</p

Comparison of gene expression in human H1 ES-DBCs and mature beta-cells.

<p>Expression of the top-ten most significantly enriched mRNAs in either adult mature or fetal beta-cells as described by <i>Hrvatin et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164457#pone.0164457.ref026" target="_blank">26</a>]</i> were examined in ES-DBCs vs. the human adult islets via real time RT-PCR assay. (*<i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, unpaired two-tailed <i>t</i>-test, n = 3).</p

The mRNA expression analysis of pancreatic islet, beta-cell and related genes in the differentiated human H1 ES-DBCs.

<p>(A) Exact copy number of insulin mRNA molecules in the ES-DBCs and human islets by digital droplet RT-PCR (GAPDH was used for normalization). Quantitative real time RT-PCR analysis for (B) endocrine hormones, (C) Chromogranin A, (D) pancreatic transcription factors, Ca⁺² and K⁺ channels genes, (E) Glucose transporters (<i>GLUT1 and 2</i>) and <i>PCSK2</i> as the enzyme required for pro-insulin processing and in the ES-DBCs compared to human islets. (*<i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, unpaired two-tailed <i>t</i>-test, n = 3).</p

The efficiency of Definitive Endoderm (DE) and Gut Tube Endoderm formation at the stage 1 and 2 of the differentiation protocol.

<p>(A) Flow cytometry, and immunofluorescence staining for DE-specific markers in the differentiated H1 ES cells. (B) Quantitative RT-PCR results for Gut Tube Endoderm-specific markers are shown in (B), showing genes up-regulated in the stage 1, and (C) maintained highly expressed genes in the stage 2. Scale bar = 40μm. (*<i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, unpaired two-tailed <i>t</i>-test, n = 3).</p

Short protocol outline.

<p>(A) Schematic overview of the 25 to 30-day protocol to generate human H1 ES-derived beta-like cells (DBCs). Below, images of the differentiated H1 cells and the control cells (Non-Treated ES cell) at each stage are shown. The arrow symbol identifies tube-like structure in the differentiated cells in the stage 2. The star symbol identifies detached dead cells as spheres in the Non-Treated cells in stage 4. Scale bar = 100μm for all cell images. The <i>red font</i> indicates modifications to molecules or timing in comparison to the protocol described by Rezania et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164457#pone.0164457.ref009" target="_blank">9</a>]. (B) Expression analyses of <i>SOX17</i>, <i>FOXA2</i> and <i>Gooscoid</i> as Definitive Endoderm (DE), <i>Sox1</i> as ectoderm, and <i>Brachyury</i> as mesoderm-specific markers in the H1 ES cells differentiated on MEF, Mouse Embryonic Fibroblast; as EB (Embryoid Bodies) or on Geltrex, analyzed by quantitative RT-PCR. (* <i>p< 0</i>.<i>05</i>, **<i>p< 0</i>.<i>01</i>, p***<<i>0</i>.<i>001</i>, significant differences between the treated and control cells in each condition, unpaired two-tailed <i>t</i>-test, n = 3).</p