Effect of CTCF binding activity on interactions among <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i> gene promoters.

<p>(<b><i>A</i></b>) Detection of CTCF binding activities at sites 2 and 3 in Pax6 promoter region by ChIP based PCRs in both lentivirus-infected Lv-control and CTCF-knocked down CTCF-shRNA cells during differentiation. (<b><i>B</i></b>) Detection of decreased CTCF-binding on site 2 of <i>RCN1</i> gene promoter during differentiation of both Lv-control and CTCF-shRNA cells. (<b><i>C</i></b>) Detection of decreased CTCF-binding on site 2 of <i>ADAM17</i> gene in differentiated Lv-control and CTCF -shRNA cells. (<b><i>D</i></b>) Statistic analysis of the significant decreases in CTCF binding in promoter regions of <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i> genes in differentiated Lv-control and CTCF-shRNA cells, respectively. ChIP-based PCR was performed to amplify the selected CTCF bound DNA fragments in <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i> promoter regions, respectively. Input and CTCF AB^- experiments were performed as controls with non-immunoprecipitated chromatins and in the absence of CTCF-specific antibody, respectively. Data were obtained from six independent ChIP and PCR experiments. Symbols “*” and “**” indicate significant differences between control and differentiated cells, control and CTCF-shRNA cells and differentiated control and CTCF-shRNA cells, respectively (<i>p</i><0.05, n = 6).</p

Characterization of differentiation-induced corneal epithelial cells.

<p>(<b><i>A</i></b>) Morphological changes in culture during differentiation of HTCE cells. (<b><i>B</i></b>) AE3 and AE1 keratin panel staining in HTCE cell differentiation. (<b><i>C</i></b>) Time course of K12 mRNA expression following induced differentiation of HTCE cells. Differentiation of HTCE cells was induced by adding 1.2 mM calcium and 5% FBS in the normal culture condition up to 120 h. (<b><i>D</i></b>) Detection of K12 and p63 expression in HTCE cell differentiation by Western blot up to 120 h. (<b><i>E</i></b>) Expression of K12 and p63 mRNAs and proteins in human limbal stem/progenitor (HLS/P) and corneal epithelial (HCE) cells. Cells were grown and induced to differentiation in chamber slides and photos were taken by light microscopy. These cells were then washed in PBS and fixed with 95% ethanol before staining. Staining protocols were used as suggested by manufactures. Furthermore, RNAs were extracted from HTCE, HLS/P and HCE cells, and reverse-transcribed into cDNAs before real-time qPCR analysis was performed using K12-specific primers as indicated in Materials and Methods. Symbol “*” indicates significant differences after 72 h induction (<i>p</i><0.05, n = 6). Photos were taken by a Zeiss AXIO microscope (x20).</p

Effect of altered CTCF levels on expressions of <i>PAX6</i>, <i>RCN1</i> and <i>ADAM17</i>.

<p>(<b><i>A</i></b>) Expression patterns of CTCF, PAX6, ADAM17 and RCN1 proteins in Lv-control and CTCF mRNA knocked down CTCF-shRNA cells following a differentiation time course. (<b><i>B</i></b>) Statistical analysis of the effect of knocking down <i>CTCF</i> mRNA by <i>CTCF</i>-specific shRNA on expressions of CTCF, PAX6, ADAM17 and RCN1 proteins during HTCE cell differentiation at 48 h. (<b><i>C</i></b>) Comparison of expression in RNA levels of <i>K12</i>, <i>PAX6</i>, <i>ADAM17</i>, and <i>RCN1</i> in lentivirus-infected control and CTCF-shRNA cells following differentiation time courses. (<b><i>D</i></b>) Significant alteration of G₀/G₁ and S phases in cell cycle distribution of differentiation-induced CTCF-shRNA cells. Western blots and RT-qPCR were described in details in materials and methods. Treatments are as indicated. Cell cycle analysis was also described previously. Symbols “*” and “**” indicate the statistical significance between Lv-control and CTCF-shRNA cells before and after differentiation, respectively. Significant differences among groups were determined by One-way ANOVA and Tukey’s tests, and then Student’s <i>t</i> test was used to determine the significant difference between two samples at <i>P</i><0.05 (n = 4 to 6).</p

Correlation of CTCF and PAX6 expression during corneal epithelial cell differentiation.

<p>(<b><i>A</i></b>) Cell cycle analysis by flow cytometry revealed significantly increased and decreased cell populations in G₀/G₁ and S phases, respectively. (<b><i>B</i></b>) Time courses of CTCF and <i>PAX6</i> mRNA expressions in HTCE cell differentiation. (<b><i>C</i></b>) Detection and analysis of CTCF and <i>PAX6</i> mRNA expressions in HLS/P and HCE cells. (<b><i>D</i></b>) Western analysis demonstrated that there is an opposite expression pattern between CTCF and Pax6 following a time course of HTCE cell differentiation (Diff). Flow cytometric analysis of HTCE cells with or without differentiation was performed as described in materials and methods section. Expressions of CTCF and Pax6 in both their protein and RNA levels were detected by Western blots and quantitative real-time RT-PCR during HTCE cell differentiation for as long as 96 h after induction. Symbol “*” indicates significant differences (<i>p</i><0.05, n = 4).</p

CTCF-mediated chromatin interactions of <i>PAX6</i> with <i>RCN1</i> and <i>ADAM17</i> genes.

<p>(<b><i>A</i></b>) Illustration of circularized chromosome conformation capture (4C) primers designs relative to HindIII restriction sites which are adjacent to CTCF binding sites 2 and 3 on <i>PAX6</i> promoter. (<b><i>B</i></b>) Six product genes were listed as CTCF-mediated <i>PAX6</i> interactive genes (score >1.5) from analysis with 4C and microarray results. (<b><i>C</i></b>) Confirmation of 4C results by FISH to truly identify interaction in promoter regions of <i>PAX6</i> and <i>RCN1</i> genes in lentiviral infected control (Lv-control), and lentiviral CTCF-shRNA infected (CTCF-shRNA) and CTCF-knocked down HTCE cells. (<b><i>D</i></b>) Verification of 4C technology by FISH to truly identify interaction in promoter regions of <i>PAX6</i> and <i>ADAM17</i> genes in Lv-control and CTCF-shRNA cells. Details of 4C and use of microarray were discussed in materials and methods. FISH assays were performed as described in materials and methods to determine positions of <i>PAX6</i> gene locus relative to both <i>RCN1</i> and <i>ADAM17</i> gene loci. The BAC clones RPCI-11-26B16 encompassing <i>PAX6</i> gene was labeled with Red 5-ROX dUTP (Empire genomics). Other BACs RPCI-11-122P23 for <i>RCN1</i> and RPCI-11-257F10 for <i>ADAM17</i> were labeled with Green 5-Fluorescein dUTP (Empire genomics).</p

IGF-1Rβ phosphorylation may play an essential role in the nuclear translocation of IGF-1Rα in GD orbital fibroblasts.

<p>(<b>A</b>) NVP-AEW-541, a specific tyrosine kinase inhibitor blocks IGF-1R phosphorylation (<b>B</b>) NVP-AEW-541 blocks nuclear IGF-1Rα accumulation. GD orbital fibroblasts were treated as indicated and stained with anti-IGF-1Rα Ab (green) and counterstained with PI. (<b>C</b>) Western blot analysis of nuclear proteins from GD orbital fibroblasts treated with nothing, IGF-1, NVP-AEW-541, or the combination and probed with anti- IGF-1Rα and anti-c-jun Abs. The findings are representative of three experiments performed.</p

siRNA directed against ADAM17 attenuates the nuclear accumulation of IGF-1Rα in GD orbital fibroblasts.

<p>Fibroblasts were transfected with control siRNA or that specifically targeting ADAM 17. Some were left untreated while others were treated with IGF-1. Cells were fixed and stained with anti-IGF-1Rα Ab/Orgeon Green anti-rabbit Ab. Quantification of nuclear intensity was conducted so that each column represents the mean of ten randomly chosen nuclei ± SD. *, p<0.002 vs control; **, p<0.01 vs IGF-1. n = 3 independent determinations. (Inset) Adam17 siRNA or control siRNA was transfected into GD fibroblasts, cells were lysed, and proteins subjected to Western blot analysis by probing with anti-ADAM 17 Ab, stripping the membrane and incubating with anti-β-actin.</p

ADAM 17 appears to play a critical role in nuclear accumulation of IGF-1Rα.

<p>(<b>A</b>) GD orbital fibroblasts express ADAM 17 protein as assessed by Western blot. (<b>B</b>). TAPI-1, a specific inhibitor of ADAM17 activity, blocks the nuclear accumulation of IGF-1Rα provoked by IGF-1 in GD orbital fibroblasts. (<b>C</b>) Quantification of nuclear grey color signal intensity. Each bar in the histogram represents the mean ± SD of ten nuclei randomly chosen for each treatment group. *, p<0.05 vs IGF-1; **, p<0.01 vs control, n = 3 independent determinations.</p

IGF-1R protein differentially accumulates in the nuclei of TAO orbital fibroblasts and derives from the fibroblast surface.

<p>(<b>A</b>) Western blot analysis of nuclear and cytoplasmic IGF-1Rα in GD orbital fibroblasts before and following IGF-1 (10 nM) treatment for 16 h. Cells were subjected to subcellular fractionation as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. (<b>B</b>) Nuclear IGF-1Rα content in GD and control orbital fibroblasts before or following treatment with either IGF-1 (10 nM) or GD-IgG (15 µg/ml) for 16 hours. (<b>C</b>) Insulin fails to alter the nuclear content of IR or IGF-1Rα in GD orbital fibroblasts. Cells were treated with nothing or insulin (15 µg/ml) for 16 hrs. They were subjected to subcellular fractionation and Western blot analysis. (<b>D</b>) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. (<b>E</b>) Control and GD fibroblasts were subjected to ¹²⁵I-IGF-1 cross-linking with either the cell-impermeable agent, BS, or the cell permeable agent, DSS. They were then treated with IGF-1. Nuclei were separated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and subjected to quantification of radioactivity. Results are representative of three experiments performed.</p

Confocal imaging of IGF-1R reveals nuclear accumulation in TAO fibroblasts following treatment with either IGF-1 or GD-IgG.

<p>(<b>A</b>) untreated (control, upper panels) or IGF-1 (10 nM) (lower panels) for 16 h. reveals localization of IGF-1Rα (green) as indicated by the yellow overlay nuclei in GD fibroblasts. Chromatin was stained by PI (red). Control fibroblasts failed to respond. The images labeled IGF-1Rβ (green) demonstrate an absence of effects with IGF-1 on nuclear accumulation. Scale bar, 25 µm. (<b>B</b>) IGF-1Rα (green) co-localizes in the PI-stained nuclei (yellow overlay) following treatment with GD-IgG (15 µg/ml) in GD but not in control fibroblasts. (<b>C</b>) Dexamethasone (10 nM) attenuates the nuclear accumulation of IGF-1Rα (<b>D</b>) as does the IGF-1R-blocking mAb, 1H7 (5 µg/ml). These experiments have been performed 4 times.</p