Additional file 1 of A Boolean network model of human gonadal sex determination

Supplementary information. (PDF 47 kb

Survival analysis of women with CC according to International Federation of Gynecology and Obstetrics (FIGO) staging, CN-AG, and gene expression profiles.

<p>The Kaplan-Meier curves for FIGO staging, the whole and 3q %CN-AG, and gene expression profiles of genes involved in glycolysis and APC/C-dependent proteasomal protein catabolic process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097842#pone-0097842-g009" target="_blank">Figure 9</a>) are shown. Patients were followed up an average of 63 months. The p value was calculated by comparing the curves with the log-rank test. Censored patients are labeled with transverse lines.</p

DAVID functional annotation cluster analysis in the 2006 genes differentially expressed in cervical cancer.

<p>*The cluster number was obtained when the analysis was run with the whole gene set, including up- and down-regulated genes.</p><p>FC = Fold change is the ratio of the proportion of genes in the tested list versus the Human Gene Reference database.</p><p>NC =  No clustered in up (+) and down (−) regulated genes analyzed separately.</p><p>The clusters in italics were enriched when the functional annotation cluster analysis was run at the highest stringency, and the number inside the parenthesis indicated the order the cluster occupied in the list.</p

Analysis workflow of 59 cervical cancer cases.

<p>Figure shows the analysis workflow of the 59 CC cases explored in this study. All CC samples were HPV16 positive and were investigated with microarrays–31 for mapping CN-AG and 55 for global gene expression, with 27 CCs in common. These 27 CCs were used for the analyses of global gene expression and the correlation between the CN-AG and gene expression. For the hierarchical clustering analysis, the expression profiles of the 55 CC samples were included. Five-year survival was investigated in 55 patients, 51 explored for gene expression and 28 for CN alterations, with 24 CCs in common. See material and methods section for details of the procedures.</p

Validation of genes amplified and deregulated located in 3q by qPCR and qRT-PCR.

<p>The top panel shows the average copy number of 7 genes (<i>CLDN1</i>, <i>ECT2</i>, <i>NAALADL2</i>, <i>NLGN1</i>, <i>PLOD2</i>, <i>PLSCR1</i>, and <i>PLSCR4</i>) located in 3q explored with qPCR in controls (lymphocytes) and tumors with 2 or 3–4 copies identified with the 500 K microarray. Whiskers of each bar represent the standard error of the mean. The dotted red line shows the value for 2.5 copies calculated with qPCR (see material and methods). The bottom panel shows the correlation of gene expression of 8 genes (<i>MCM2</i>, <i>PLOD2</i>, <i>PLSCR1</i>, <i>SMC4</i>, <i>ECT2</i>, <i>NLGN1</i>, <i>RFC4</i>, and <i>CLDN1</i>) located in 3q explored in 27 tumors and 6 controls with both the HG 1.0 ST microarray and qRT-PCR techniques. Log₂ values of the normalized intensity signals obtained with the microarray (robust multichip average values) and qRT-PCR were plotted. Trend line (black line), correlation coefficient (r), and p value were calculated with Pearson’s correlation test.</p

Validation of the GeneChip Human Mapping 500(500 K) microarray with the high-density CytoScan HD microarray.

<p>Intensity signals of single nucleotide polymorphisms (SNPs) or non-polymorphic probes are expressed as log₂ ratios from chromosomes 3, 4, and 5 of the tumor R496 explored using the 500 K microarray (upper panel) and HD 2.7 microarray (lower panel). In both panels, the y-axis depicts a log₂ ratio scale from –1.5 to 1.5, and the x-axis shows the ideogram of the explored chromosomes with genome positions. The horizontal line crossing the point y = 0 corresponds to 2 copies. The average density of explored positions is more than 5 times higher in the HD 2.7 microarray than that in the 500 K microarray (see materials and methods).</p

Amount of copy number (CN)-altered genome and frequency of recurrent CN-altered genes.

<p>Panel A shows box plots with the distribution of tumors (n = 31) according to the percentage of total, deleted, or amplified CN-altered genome (CN-AG). Panel B shows the distribution of tumors, grouped as low (n = 11), medium (n = 10), and high (n = 10) according to the percentage of global and 3q CN-AG. The horizontal lines inside the boxes represent the median (solid) and average (dotted), and the whiskers represent the minimum and maximum values within the 1.56 interquartile range from the end of the box. Values outside this range are represented by black circles. The decline in the accumulated frequency of recurrent CN-altered genes, as increase the number of tumors that shared the same altered gene, is shown for the whole genome (Panel C) or for chromosomes with significant high %CN-AG (Panel D). Combined genes are those that were deleted in some and amplified in other tumors.</p

DAVID functional analysis of genes correlated and non-correlated with 3q amplification in high-CN tumors.

<p>See the legends in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097842#pone-0097842-t003" target="_blank">Table 3</a>.</p

Identification of CN-altered genes in biological processes enriched in high-CN tumors.

<p>Shown are the numbers of 2-copy or CN-altered genes in ≤3 tumors (green bars) and CN-altered genes in ≥4 tumors (blue bars) among the biological processes enriched in the subset of genes deregulated exclusively in high-CN tumors.</p

Patients followed up on average for 63 months for survival evaluation.

a<p>ACC, Adenocarcinoma. SCC, Squamous Cell Carcinoma. ASCC, Adenosquamous Cell Carcinoma.</p><p>bHT, Radical Hysterectomy. Tele, teletherapy. Brachy, brachytherapy. Chemo, chemotherapy with Cisplatin. i. Means incomplete treatment.</p>c<p>Status alive at the last follow up record and death was caused by primary tumor of cervical cancer, except the case labeled with an asterisk. The cause of death was unknown.</p>d<p>CN indicate the samples analyzed for CN (500 K array), CN/EX indicate the samples analyzed for CN (500 K array) and gene expression (HG 1.0 ST array), and EX indicate the samples analyzed for gene expression (HG 1.0 ST).</p