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

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 One +dox and one—dox mice were withdrawn from the study after one month.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

<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

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

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

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

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

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