MIG-6 protein levels in lung cancer and melanoma cell lines.

<p>Whole cell lysates were prepared from the indicated cell lines, and MIG-6 was determined by western blot analysis using anti-Mig-6 polyclonal antibody. As a loading control, the same blot was probed with anti- β-actin antibody.</p

Working models for epigenetic regulation of <i>MIG-6</i> expression by 5-aza-dC and TSA.

<p>(A) In lung cancer cells, TSA might indirectly regulate <i>MIG-6</i> through two mechanisms. The first could be that TSA inhibits histone deacetylation in the promoter of gene <i>X</i>, resulting increased production of X protein which enhances <i>MIG-6</i> gene expression by associating with the TSA-response element in exon 1. The second might be that direct acetylation of protein X influenced by TSA results in increased transcription of <i>MIG-6</i>. (B) In melanoma cells, the regulation of <i>MIG-6</i> expression by 5-aza-dC might be direct or indirect. 5-Aza-dC might inhibit DNA methylation in the promoter of gene <i>Y</i>, leading to up-regulation of its product and thus indirectly enhancing <i>MIG-6</i> expression; or it might directly inhibit DNA methylation outside the tested 1.383-kb <i>MIG-6</i> promoter regulatory region, allowing easy access of a transcriptional co-activator to enhance <i>MIG-6</i> expression. “P” indicates the promoter of each gene; “E1” indicates exon 1 of the <i>MIG-6</i> gene.</p

<i>EGR1</i> displays an expression pattern similar to that of <i>MIG-6</i> in lung cancer and melanoma cell lines upon 5-aza-dC and TSA treatment.

<p>The expression of <i>EGR1</i> was determined by RT-PCR. (A) TSA induced <i>EGR1</i> expression in the NCI-H226, NCI-H522, NCI-H596, and A427 lung cancer cell lines. (B) 5-Aza-dC induced <i>EGR1</i> expression in the M14, MALME-3M, SK-2, SK-MEL-28, and UACC-257 melanoma cell lines. <i>GAPDH</i> expression was used as an internal control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038955#pone-0038955-g003" target="_blank">Figure 3</a>).</p

The <i>MIG-6</i> promoter is hypomethylated in lung cancer and melanoma cell lines.

<p>The <i>MIG-6</i> promoter was amplified from bisulfite-converted DNA and cloned into a TOPO TA-cloning vector. The status of <i>MIG-6</i> promoter methylation in (A) lung cancer cell lines and (B) melanoma cell lines was evaluated by sequencing 10 randomly picked colonies from each line for methylated cytosine residues. Each red bar represents a CpG site. The open ovals indicate unmethylated CpG sites and the solid ovals indicate methylated sites. EBC-1 cell line was used as a negative control to show the basal methylation status of MIG-6 promoter.</p

The <i>MIG-6</i> promoter is not affected by histone deacetylation.

<p>The cells were treated with or without TSA (1 µM) for 24 h, and a ChIP assay was performed using anti-acetyl histone H3 antibody. As a negative control, normal rabbit serum was used for immunoprecipitation. The DNA fragments cross-linked and co-immunoprecipitated with the acetylated histone H3 were purified and used for PCR amplification of the promoters of <i>MIG-6</i> and <i>GAPDH</i>. EBC-1 cell line was used as a negative control to show the basal histone deacetylation status of MIG-6 promoter.</p

Induction of <i>MIG-6</i> expression by 5-aza-dC and TSA is regulated at transcriptional level.

<p>Total RNAs were isolated from cells treated with or without 5-aza-dC (10 µM) and/or TSA (1 µM), and <i>MIG-6</i> expression was determined by RT-PCR analyses. <i>GAPDH</i> expression was used as an internal control. (A) TSA increased <i>MIG-6</i> mRNA in the four lung cancer cell lines. (B) <i>MIG-6</i> mRNA in the five melanoma cell lines was increased by 5-aza-dC.</p

Dissecting the minimal TSA-response element in <i>MIG-6</i> by mutational analysis.

<p>(A) The sequences of P(−76/+50) that contain the <i>MIG-6</i> promoter and the minimal TSA-response element are shown. For each mutant reporter construct (m3-m11), the underlined sequence was mutated to GAATTC. (B and C) A luciferase reporter assay was performed in A427 lung cancer cells by transiently transfecting the indicated reporter plasmid with or without TSA treatment. The error bars represent standard deviation and all assays were performed in triplicate.</p

Increased <i>Lrp6</i> expression in basal-like human breast cancer.

<p>The relative expression of <i>Lrp6</i> in breast cancer samples analyzed by Affymetrix and organized into defined subgroups. Each dot represents the relative expression of <i>Lrp6</i> in one tumor sample. (A) Expression data was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005813#pone.0005813-Richardson1" target="_blank">[30]</a>. <i>Normal</i> includes samples obtained from normal breast tissue. (B) Expression data was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005813#pone.0005813-Herschkowitz1" target="_blank">[31]</a>. <i>Normal Breast-like</i> includes samples obtained from normal and cancerous breast tissue that exhibited expression profiles similar to that of normal breast tissue. A subset of breast cancers within the basal-like subgroup of both studies exhibit increased expression of <i>Lrp6</i>.</p

<i>Lrp6</i> is required for embryonic mammary development.

<p>(A) Carmine-stained skin pads of inguinal mammary glands at E18.5. While in the <i>Lrp6^+/+</i> mammary gland the nipple, rudimentary ductal tree, and fat pad are all normally developed, the <i>Lrp6^−/−</i> mammary gland contains a small nipple, a single ductal out-growth, and an abnormally small fat pad. <i>Dashed lines</i> indicate inguinal epithelium. (B) Oil red O staining of mammary fat pads from <i>Lrp6^+/+</i> and <i>Lrp6^−/−</i> embryos. The <i>Lrp6^−/−</i> fat pad is abnormally small compared to that of the littermate control. <i>Scale bar</i>: 0.5 mm. (C-D) X-gal-stained <i>BAT-gal</i> embryo whole mounts (C) and histology sections of mammary placode (D) at E12.5. Cells expressing BAT-gal are stained blue. (C) X-gal stains the mammary placodes of <i>BAT-gal;Lrp6^+/+</i> embryos dark blue. <i>Arrow heads</i> indicate mammary placodes number 2, 3, 4, and 5. Mammary placodes are not readily visible on X-gal-stained <i>BAT-gal;Lrp6^−/−</i> embryo whole mounts. (D) On the histological level, the mammary placodes of <i>BAT-gal;Lrp6^−/−</i> embryos are significantly smaller and exhibit fewer cells with <i>BAT-gal</i> expression than the mammary placodes of littermate controls. <i>Dashed lines</i> indicate inguinal placodes.</p

Haploinsufficiency for <i>Lrp6</i> in postnatal mammary development.

<p>(A) Representative mammary whole-mount preparations are shown for juvenile (5-week-old) mice. The <i>arrows</i> indicate typical terminal end buds; <i>LN</i>, lymph node. (B) The result of morphometric analysis of the average number of TEBs at 5 weeks and (C) of branches per gland at 11 weeks. At least 10 animals of each genotype were analyzed for each time point. In the absence of one copy of <i>Lrp6</i>, the number of TEBs is reduced by 33% (<i>p</i> = 1.3×10⁻⁶, 2-tailed <i>t</i> test assuming unequal variances), and the number of branches per gland is reduced by 17% (<i>p</i> = 8.4×10⁻³, 2-tailed <i>t</i> test assuming unequal variances) compared with <i>Lrp6</i>^+/+ littermate controls. (D) Mammary whole mounts containing ductal colonization originating from transplanted <i>Lrp6^+/+</i> or <i>Lrp6^+/−</i> mammary epithelial cells. Also shown is a transplantation control whose inguinal fat pads were cleared of endogenous epithelium but not injected with mammary cells. (E) The inguinal mammary gland from an adult <i>Lrp6^+/−;Lrp5^−/−</i> female. The mammary gland contains a fat pad and a nipple with associated epithelium but lacks a ductal tree. <i>Box</i> indicates the nipple epithelium.</p