<i>CXCR4</i> expression analysis using semi-quantitative RT-PCR in breast cancer tumor cell lines and <i>CXCR4</i> expression after 5-aza-2′-deoxycytidine (D-Aza) treatment.

<p>(A) The bands represent <i>CXCR4</i> expression in the PMC-42, MCF7, MDA-MB-436 cell lines and (B) MDA-MB-435 mock or MDA-MB-435 D-Aza represent the MDA-MB-435 cell line before and after treatment with 5-aza-2′-deoxycytidine, respectively. The <i>GAPDH</i> gene was used as a positive control in both experiments. MW, Molecular Weight, NC represents the PCR reaction without DNA (negative control).</p

MSP analysis in breast cancer cell lines and primary breast tumors.

<p>(A) Primer standardization for methylated and unmethylated conditions in tumor cell lines. (B) MSP analysis of primary tumors. Thirteen samples are represented. MW, Molecular Weight; NC, Negative Control.</p

Kaplan Meier curves for overall survival and disease-free survival according to the methylation status of <i>CXCR4</i> and <i>CXCL12</i>.

<p><i>CXCR4</i> methylation status and the correlation with (A) overall survival (OS) and (B) disease-free survival (DFS) are shown. <i>CXCL12</i> methylation status and association to <i>CXCR4</i> methylation for (C) OS and (D) DFS are shown.</p

Sequence of the primers used for RT-PCR, nested-PCR and MSP.

<p><b>Abbreviations:</b> M, specific for methylated condition; U, specific for unmethylated condition.</p

Clinicopathological features of 69 patients with primary breast carcinomas and methylation status of <i>CXCR4</i> gene.

<p><b>Abbreviations:</b><i>p</i>, value from statistical analysis <i>χ²</i> test and Fisher's exact test; M, methylated; U unmethylated; significant data are in bold.</p><p>*<i>CXCL12</i> and <i>ESR1</i> methylation data were used from a previous study published by our group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029461#pone.0029461-Ramos1" target="_blank">[17]</a>.</p

Bisulfite sequencing of the <i>CXCR4</i> gene promoter in the breast cancer cell lines.

<p>The cell lines used are shown. The nineteen dinucleotides are numbered in agreement with the sequence. The open circles represent the unmethylated dinucleotides while the gray to black portion represents the percentage of methylation. On the right side methylation pattern are represented according to data of RT-PCR and the absolute percentage value. The arrows below the CpG dinucleotides represent the MSP primers that were used.</p

Wound healing assay in MCF7 cells.

<p>A<b>:</b> Representative images from mock and FN-treated cells were shown. The scratched cells in a line had images obtained under un inverted optical microscopy (20X). B<b>:</b> The graphic represents % of wound-closure after 60 h in culture. Statistical comparison was performed using Student’s <i>t</i> Test. *<i>p</i><0.05.</p

<i>MMP2</i> expression after treatment in breast cancer cell lines.

<p>A: The expression of <i>MMP2</i> after treatments in MCF7 cells was shown. Mock (blue); 5-Aza-treated (white), FN-treated (red) and recultured (green). Results were expressed as mean S.E.M. and statistical comparison was performed using <i>t test</i> analysis. *<i>p</i><0.05 when compared to mock; #<i>p</i><0.05 when FN-treated compared to recultured. B: The expression of <i>MMP2</i> in MDA-MB-436 cells, mock and after FN treatment were shown. Mock (blue) and FN-treated (orange) were expressed as mean S.E.M. and statistical comparison was performed using Student’s <i>t</i> Test. **<i>p</i><0.05 when compared to mock.</p

Epigenetic changes in the <i>MMP2</i> gene promoter in MCF7 cells.

<p>A: Sequencing of the <i>MMP2</i> gene promoter. Closed and open circles represent methylated or unmethylated CpGs, respectively. On the left the number represent the sequenced clones. The 49 analyzed CpGs in the <i>MMP2</i> promoter region are shown. Mock cells are at the top, above is 5-Aza-treated, hereafter FN-treated, recultured (MCF7 FN-treated and then cultured for 48 hours without fibronectin). The global methylation percentage is also shown at the right. B: Graphical analysis of CpG methylation pattern in the <i>MMP2</i> promoter gene. The percentages of CpGs that were methylated in MCF7 mock (blue), MCF7 FN-treated (red) and MCF7 recultured (green) were shown. C: ChIP quantitative PCR analysis. Ct values were normalized between target and endogenous control (<i>MMP2</i>/<i>GAPDH</i>) and the results of mock (blue), FN-treated (red) and recultured (green) cells are shown. At the bottom the samples are separated according to the antibody used in the immunoprecipitation. Statistical comparison was performed using Student’s <i>t</i> Test. *<i>p</i><0.05.</p