Additional file 2: Figure S2. of Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis

Comparable E6 mRNA expression levels in mouse skin. Skin biopsies from K14-HPV8-E6wt and K14-HPV8-E6K136N lines were taken at the indicated time points after UV irradiation and HPV8 E6 mRNA levels were measured in duplicate by qRT-PCR and normalized to the mRNA levels of HPRT1 (n = 6; untreated skin, p = 0.5414; 3d post UV-treatment, p = 0.2904). Data are presented as mean ± SEM. (PPTX 59 kb

Additional file 1: Figure S1. of Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis

Inhibition of T^T repair by β-PV E6. HT1080 cells expressing E6 genes of β-PV types 5, 8 and 20 were irradiated with UVB and levels of T∧T were assayed using In-Cell Western analysis (n = 4 in duplicate, HPV5,****, p < 0.0001; HPV8, ****, p < 0.0001; HPV20, ***, p = 0.0002). Data are presented as mean ± SEM. (PPT 151 kb

Galectin-3 prevents TCR synapse formation in late-stage T cells.

<p>(A) CD7⁻ and CD7⁺ T cells were activated by incubation with anti-CD3 mAb plus anti-human CD28 mAb or as control by an isotype-matched IgG1 (medium). Cells were stained for TCR-alpha/beta (green), CD7 (blue), CD3 (red) and for galectin-3 (yellow). Immunofluorescence was visualized by a LSM. Alternatively cells were stained with mAbs specific to CD7 and subsequently recorded for gal-3 expression by staining with the anti-human gal-3mAb or as control by an isotype-matched IgG and analyzed by flow cytometry. A representative experiment out of five is shown. Galectin-3 specific signals were quantitatively recorded by a LSM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells for each data point was recorded. (B) To monitor location of galectin-3 in lipid raft formation, isolated CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine for 24 hrs and then activated for various time intervals (1 min until 20 min) on coverslips coated with the agonistic anti-CD3 mAb plus anti-CD28 mAb. T cell stimulation was stopped with paraformaldehyde and cells were stained with anti-TCR-alpha/betamAb (blue), anti-CD7 mAb (red) and anti-galectin-3 mAb (yellow) together with CtxB (green) for lipid rafts staining. Immunofluorescence was visualized by a LSM and quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. T cell staining after 5 min stimulation of one representative experiment out of five is exemplarily shown. (C) CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine and activated as described in (B). Frequencies of cells producing IFN-gamma and showing degranulation indicated by CD107a was monitored by flow cytometry. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#pone-0030713-g003" target="_blank">Fig. 3</a> represent the mean ± SEM of five experiments and were compared using a paired t-test. * p<0.05. FU: fluorescence unit; MFI: mean fluorescence intensity.</p

TCR synapse formation is impaired in late-stage T cells.

<p>CD8⁺ CD45RO⁺ T lymphocytes were incubated on coverslips coated with the agonistic anti-CD3 mAb (UCHT-1) plus anti-CD28 mAb (CD28.2) or with the anti-CD2 mAbs (L303.1) and (L304.1) plus the anti-CD28 mAb (CD28.2). Cells were activated for various time intervals (1 min until 20 min), incubation was stopped by addition of paraformaldehyde, and cells were stained with TCR-alpha/betamAb (green) plus CD7 mAb (blue) or alternatively with CD2 mAb (blue) plus CD7 mAb (green) together with cholera toxin B (CtxB) (white) for lipid raft staining. Cells were analyzed on a LSM 510 with 630× microscope magnification. A minimum of 100 cells for each data point was recorded. Representative images after 0 min, 1 min, 5 min, 10 min and 20 min stimulation out of five independent experiments are shown. Synapse formation intensity (CTxB intensity) was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells of each cell population on each coverslip was recorded. Data represent mean scores from five experiments ± standard error of the mean and were compared using a paired t-test. * p<0.05. FU: fluorescence unit.</p

Reconstruction of novel transcripts by <i>de-novo</i> assembly.

<p>Histograms display lengths of reconstructed sequence contigs assembled from unmapped reads of NB2 stage 4 and stage 4S samples (y-axis in log-space). Two independent assembly methods, Trinity and Oases, were used in the reconstruction. The grand total number of contigs reconstructed within each assembly is displayed in the rightmost column. Reconstructed contigs are annotated with their putative taxonomic origin as inferred by comparison with NCBI nucleotide (nt) and protein (nr) archives using TBLASTX database searches.</p

Estimation of required cellular transcript abundances for achieving a given transcript coverage.

<p>Sequencing coverage of viral transcripts is depending on the average number of transcript copies per cell in the sequenced sample, on the length of the viral transcript being sought, and on characteristics of the sequencing process. In order to better visualize the optimal sequencing depth required for detection of viral factors, we estimated the required number of transcript copies per cell for different sequencing depths. These sequencing depths are expressed as factors relative to the depths employed for the NB1/NB2 panel generated in this study (which are here reported as a relative sequencing depth of 1).</p

Sequencing panel characteristics.

<p>Sequencing characteristics of neuroblastoma (NB), positive control (POS), and negative control (NEG) panels.</p

Virana's approach to identifying viral transcripts in human tumors.

<p>a) Transcriptome sequence samples are first mapped to a combined set of human and viral reference sequences in a splicing-aware fashion. b) Unmapped or discordantly mapped read pairs are further processed by assembly methods to detect novel viruses or transcript chimeras that may indicate proviral integration events. c) Reads mapping to one or more viral genomes (HITs) are analyzed in an integrated fashion by considering human homologous mapping locations and viral taxonomies. This process results in a number of homologous regions (HOR) for each viral family. HORs are represented as multiple sequence alignments incorporating a wealth of sequence information. Alignments are further enriched by taxonomic annotations and phylogenetic analyses.</p

Detection of divergent viruses.

<p>Performance comparison of Virana, CaPSID, and RINS at detecting viral reads at different rates of simulated sequence divergence among a background set comprising human genomic reads. The background set without any spike-ins of viral reads serves as negative control. Left panel: stacked bars represent absolute numbers of detected reads grouped by sequence divergence, correctness of classification (TP: true positive, FP: false positive), and detection method. Falsely classified reads not assigned to any of the viral families present in the validation are labeled as false positives (FP). Colored segments indicate to which viral families the reads were assigned. Each condition allowed for the correct detection of up to reads. Right panel: color coded markers for each condition and detection method indicating which viral families were identified. A maximum number of viral families could be correctly identified in each condition.</p

Overview of identified homologous regions in positive and negative experimental controls.

<p>Left panel: cumulative numbers of reads assigned to viral taxonomic families (log-scale). Each bar represents a homologous group (HOG) colored according to viral taxonomic family. Bars comprise several segments, each representing a homologous region (HOR). Heights of segments indicate the putative origin of reads assigned to this region (human, viral, or ambiguous). Viral families of bacteriophages are marked accordingly. Right panel: Analogous to left panel, but the lengths of bars represent relative rather than absolute abundances quantified in cumulative reads per million reads mapped (RPMM).</p