Table_1_Deep Sequencing Revealed a CpG Methylation Pattern Associated With ALDH1L1 Suppression in Breast Cancer.DOCX

<p>Hypermethylation of promoter CpG islands is generally recognized epigenetic mechanism responsible for gene silencing in cancer. However, molecular details on how this epigenetic mark triggers the process of gene downregulation are still elusive. Here, we used deep bisulfite sequencing and qPCR analysis to investigate the pattern of CpG methylation of ALDH1L1 promoter region and its association with the gene expression level in 16 paired breast cancer (BC) samples of different clinical stages. Expression of ALDH1L1 gene was suppressed in all examined BC samples up to 200-fold, and average hypermethylation level of the promoter region correlated positively with ALDH1L1 downregulation. We determined the role of every individual CpG site within the ALDH1L1 promoter, including upstream untranscribed region, first untranslated exon, and the start of the first intron, in aberrant gene expression by correlation analysis. The search revealed CpG sites which methylation has the highest impact on intensity of gene transcription. The majority of such CpG sites are located in a compact region in the first intron of the ALDH1L1 gene. These results assist in unraveling of dynamic nature of CpG promoter hypermethylation as well as demonstrate the efficiency of deep bisulfite sequencing in search for novel epigenetic markers in cancer.</p

Inhibition of tumor growth by <i>SEMA3B</i> re-expression.

<p>The growth rate of U2020 cells (U7111 clone) in SCID mice: blue line—U2020 cells without <i>SEMA3B</i> expression (+ doxycycline, 4 mice), red and yellow line—U2020 cells with <i>SEMA3B</i> expression (- doxycycline, 4 mice and 1 mouse respectively). *—no expression of <i>SEMA3B</i> gene according to the Northern blot (data not shown). One +dox and one—dox mice were withdrawn from the study after one month.</p

Methylation profile of the promoter CpG-island of the <i>SEMA3B</i> gene in lung (A) and renal (B) cancer cell lines and primary tumors.

<p>Bisulfite sequencing data, 16 CpG-dinucleotides (2–17) of the CpG-island are given. Grey squares show methylated CpG-dinucleotides, white squares—unmethylated. Numbers of primary tumors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.s001" target="_blank">S1 Table</a>. The bold numbers of CpG-dinucleotides (3–4 and 9–12) indicate the location of the primers that were used for MSP method.</p

Absence of <i>SEMA3B</i> expression in tumors grown <i>in vivo</i>.

<p>Electropherogram of multiplex PCR from plasmids, clones and SCID mice tumors of three genes. M—marker, 1—PCR from plasmid pETE/<i>SEMA3B</i>, 2—PCR from plasmid pETE/<i>TUSC2</i>, 3—PCR from plasmid pETE/<i>ZMYND10</i>, 4—PCR from U7111/<i>SEMA3B</i> cell clone 1, 5—PCR from U7111/<i>TUSC2</i> cell clone 3, 6—PCR from U7111/<i>ZMYND10</i> cell clone 4, 7—mixed cell clones, 8—PCR from tumor 1, 9—PCR from tumor 2, 10—PCR from tumor 3, 11—negative control.</p

<i>SEMA3B</i> gene expression level (A), copy number (C) and methylation status of its two CpG-islands (B) in the same ccRCC samples.

<p>Semi-quantitative PCR (A, C) and MSP (B) data. Numbers of primary tumors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.s001" target="_blank">S1 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.g005" target="_blank">Fig 5B</a>. (A) Light grey columns—samples without metastases, dark grey columns—samples with lymph node or distant metastases. (B) 1-st CpG—promoter CpG-island, 2-nd CpG—intronic CpG-island. Grey squares show methylated CpG-islands, white squares—unmethylated. (C) Grey squares show hemi- or homozygous deletions of the 5’Sema5 marker, black—amplification, white squares—retention. Assessed mean values ± error bars are represented in the “A” part.</p

Pathological and histological characteristics of the tumors.

<p>Note: The slash separates the number of samples used in the methylation studies and the expression or copy number studies by semi-quantitative RT-PCR and the number of samples used in the qPCR expression studies.</p><p>Pathological and histological characteristics of the tumors.</p

<i>In vitro</i> growth of U2020 cells (U7111 clone) depends on the expression of <i>SEMA3B</i>.

<p>A—The growth rate of U2020 cells: dashed line with squares—U7111 clone with pETE/<i>SEMA3B</i>, solid line with circles—U2020 cells with pETE (control); B—colony formation assay; C—PI-FACS analysis of cells with and without expression of <i>SEMA3B</i>. Mean values ± standard deviations for 4 replicates are represented in each case.</p

Relative mRNA level of the <i>SEMA3B</i> gene in NSCLC (A) and ccRCC (B).

<p>QPCR data, additional samplings. Light grey columns—samples without metastases, dark grey columns—samples with lymph node or distant metastases. The numbers of primary tumors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123369#pone.0123369.s001" target="_blank">S1 Table</a>. Mean values ± standard deviations for 3 replicates are represented.</p

Correlations between the frequency of CpG-island methylation and tumor stage or grade.

<p>Note: Based on MSP data. 1-st CpG—promoter CpG-island; 2-nd CpG—intronic CpG-island; <i>r</i>_<i>s</i>—Spearman’s rank correlation coefficient; <i>P</i>—p-value.</p><p>Correlations between the frequency of CpG-island methylation and tumor stage or grade.</p

Frequency and mRNA level changes of the <i>SEMA3B</i> gene in NSCLC (ADC and SCC) and ccRCC in groups of samples with different pathological and histological characteristics.

<p>Note: QPCR data. <i>R</i>—relative mRNA level.</p><p>*—in parentheses a range of mRNA level changes is shown.</p><p>Frequency and mRNA level changes of the <i>SEMA3B</i> gene in NSCLC (ADC and SCC) and ccRCC in groups of samples with different pathological and histological characteristics.</p