QPCR mRNA expression profile of <i>HYAL1</i> and <i>HYAL2</i> in SCC and RCC biopsies.

<p>The Y axis indicates the values of relative expression level of target genes in log₁₀ scale in tumour samples relative to control normal samples normalized to the reference gene <i>GAPDH.</i> The X axis shows the cDNA samples isolated from tumours at different stages (I–III). Open bars show HYAL1 and hatched bars HYAL2 expression.</p

Effect of <i>HYAL1</i> and <i>HYAL2</i> transgenes on colony formation efficiency in KRC/Y and U2020 cells compared to empty pETE vector (negative control) and wild type or mutated <i>FUS1</i> transgenes.

<p>Graphical representation summarizing three independent experiments and photographic images of Petri dishes stained with methylene blue. Values are the mean ±s.d. of three separate experiments each calculated from triplicate plates.</p

CyQUANT Cell Proliferation Assay.

<p>Effect of expression of <i>HYAL1</i> transgene in KRC/Y (A) and in U2020 (B) cells. The same is shown for <i>HYAL2</i> (KRC/Y in C and U2020 in D). Experiments were done in triplicates in the absence of doxycycline. The same experiments were done in the presence of doxycycline and showed similar results (data not shown). Plotted data points represent averages of triplicate samples, the plotted line is a linear regression fit of all data points. The assay is designed to produce a linear analytical response from at least 100–20,000 cells per well in most cell lines.</p

Analysis of <i>HYAL1</i> and <i>HYAL2</i> stably transformed KRC/Y clones.

<p>Northern analysis (A) of tetracycline regulated clones. (+), tetracycline (Tet) or doxycycline is present, gene is OFF. (−), tetracycline or doxycycline is absent, gene is ON. Growth inhibition of KRC/Y cells with <i>HYAL1</i> (B) and <i>HYAL2</i> (C) transgenes <i>in vitro</i>. Tumour growth inhibition of KRC/Y cells by <i>HYAL1</i> and <i>HYAL2 in vivo</i> in SCID mice (D). Mice were drinking water with tetracycline (+Tet, gene is OFF) or without (−Tet, gene is ON) but for simplicity curves are shown only for mice when genes were ON (no tetracycline).</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

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

<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

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