Effect of EGF and UV stress on CTCF activity.

<p>(A) Time course of UV stress-induced changes in Bcl-3 and CTCF activity. (B) Quantitative detection of UV stress-induced effect on CTCF mRNA expression by real time PCR. (C) Effect of EGF on Bcl-3 and CTCF activity following a time course. (D) Quantitative detection of EGF-induced effect on CTCF mRNA expression by real time PCR. Proteins and RNA were isolated from HCE cells at indicated time-point before/after EGF (20 ng/ml) and UV irradiation (42 µJ/cm²), respectively. Symbol “*” indicates significant differences between control and UV stress-induced cells (<i>p</i><0.05, n = 3).</p

UV stress-induced activation and interaction of Bcl-3 and p50.

<p>(A) Immuno-coprecipitation of Bcl-3 and p50 pulled down by anti-Bcl-3 antibodies. (B) Immuno-coprecipitation of Bcl-3 and p50 pulled down by anti-p50 antibodies. (C) Nuclear immuno-colocalization of Bcl-3 and p50 in UV stress-induced HCE cells. Arrows indicate activated p50, Bcl-3 and p50+Bcl-3 localized in the nucleus. (D) Statistical significance of immuno-colocalized nuclear Bcl-3 and p50 in UV stress-induced HCE cells. Bcl-3 and p50 in control, EGF stimulated and UV stress-induced HCE cells were detected by immunostaining experiments with specific antibodies against Bcl-3 and p50. Cell nuclei were detected by DAPI staining. Arrows are indicating immune activities that by were imaged by using a Nikon fluorescent microscope at 40×, and data were analyzed by Nikon software. Symbol “*” indicates significant differences (<i>p</i><0.05, n = 26).</p

Effects of EGF- and UV stress-induced activation of NF-κB pathways.

<p>(A) Time course EGF-induced phosphorylation of IκBα. (B) Time course EGF-induced degradation of IκBα. (C) Effect of UV stress on phosphorylation of IκBα. (D) Effect of UV stress on degradation of IκBα. (E) Effect of EGF stimulation on nuclear activities of p50, p65 and Bcl-3. (F) UV stress-induced nuclear activities of p50, p65 and Bcl-3. HCE cells were synchronized by serum-depletion for 24 h. Total and nuclear proteins were extracted following stimulation at indicated time points and detected by Western analysis. Symbol “*” indicates significant differences between control and induced HCE cells (<i>p</i><0.05, n = 3).</p

Effects of knocking down Bcl-3 on UV stress-induced suppression of CTCF.

<p>(A) Time-dependent effect of knocking down Bcl-3 on UV stress-induced suppression of CTCF detected by Western analysis. (B) Time-dependent effect of knocking down Bcl-3 on UV stress-induced inhibition of CTCF mRNA expression by RT-PCR. (C) Quantitative detection of UV stress-induced CTCF mRNA suppression in Bcl-3 knocking down cells by real time PCR. (D) Effect of knocking down Bcl-3 on UV stress-induced suppression of CTCF promoter activity. Data were plotted as Mean±SE and statistical significance was determined at <i>p</i><0.05 (n = 3 to 6).</p

Interaction of Bcl-3 and p50 with CTCF promoter.

<p>(A) EGF- and UV stress-induced interactions of Bcl-3 and p50 with CTCF promoter detected by ChIP assays. (B) Analysis of interactions between Bcl-3/p50 and CTCF promoter. (C) Effect of over-expression of Bcl-3 and p50 on activities of wildtype and deletion mutant of CTCF promoter. (D) Dose-response relationship between over-expression of Bcl-3 and suppression of CTCF expression. HCE cells were transfected with full-length cDNAs encoding Bcl-3 (a generous gift from Dr. Shin-Ichiro Takahashi at the University of Tokyo) and p50, CTCF reporter and the mutant CTCF reporter with κB site-deletion by lipofection. Symbol “*” indicates significant differences between control and transfected cells (<i>p</i><0.05, n = 4).</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

Methylation status of CTCF binding region in <i>Pax6</i> promoter during ES cell differentiation to radial glial cells.

<p><b><i>(A)</i></b> Detecting changes of DNA methylation in CTCF binding sites during ES cell differentiation and EB formation. <b><i>(B)</i></b> Statistical analysis of DNA methylation changes in CTCF binding motifs during ES cell differentiation and EB formation. The genomic DNA harvested from ES cells and EB cells at different stages was treated with sodium bisulfite followed by PCR of the target sequence to generate the template for Ms-SNuPE assays. Methylated cytosines at site 1–3 were revealed by ^[32]P-dCTP incorporation. Symbol “*” indicates the statistical significance determined by Student's t test at <i>p</i><0.05 (n = 3).</p

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

Identification of methylation sites within CTCF binding motifs.

<p><b><i>(A)</i></b> Putative methylation sites in the region of CTCF binding motifs upstream from <i>Pax6</i> promoter. <b><i>(B)</i></b> Quantitative determination of methylation status at cytosines in CTCF binding sties after inductions of demethylation and methylation. Genomic DNA is treated with sodium bisulfite followed by PCR of the target sequence to generate the template for Ms-SNuPE assays. Methylation statuses were evaluated by the radio of radial incorporation of ^[32]P-dCTP (representing methylated cytosine) and ^[32]P-dTTP (representing unmethylated cytosines). DNA methylations were detected by using bisulfite sodium modifications and PCR. Demethylation and methylation were induced by 5-azadCyd (1 µM) and by over-expression of Dnmt3a, respectively. <b><i>(C)</i></b> Statistical analysis of methylation percentages in site1–3. Data was shown as mean ±S.E. and represented results from three independent Ms-SNuPE assays. Symbol “*” indicates significant differences comparing with cells without treatment. (<i>p</i><0.05, n = 3).</p