Additional file 4: of Evolutionary analysis across mammals reveals distinct classes of long non-coding RNAs

Excel file of evolutionary metrics of all lncRNAs found to be conserved to the human/chimp/rat/mouse ancestor. (XLSX 19 kb

Additional File 1:

Supplementary figures and tables. (PDF 3.85 MB

Treatment with NKp30-Ig reduces PC3/<i>Luc</i> tumor growth.

<p>Male nude mice were injected with PC3/<i>luc</i> tumor cells (15×10⁶) into the SC left flanks. Three weeks after tumor implantation, mice were injected (i.p.) every second day over a period of one month with 4mg/kg of NKp30-Ig (<i>n</i> = 16) (a), NKp46D2-Ig (<i>n</i> = 9) (b) or PBS (<i>n</i> = 8) (c). Tumor progression was monitored by measuring light emission from each individual mouse in the initiation (‘start point’) and in the end (‘end point’) of the treatment period. Y-axes represent the relative (in percentage) changes in tumor size after treatment, as calculated from the integrated light emission measured in each time point (indicated numbers above columns). Shrinkage of the tumor by 20% or below its original size was referred as ‘efficient treatment’. This figure is a summary of two experiments and includes all the mice that were tested.</p

NKp30-Ig treatment reduces growth of prostate cell line DU145 <i>in vivo</i>.

<p>Male nude mice injected with the human prostate tumor line DU145 (4×10⁶), were treated with NKp30-Ig (<i>n</i> = 10), NKp46D2-Ig (<i>n</i> = 9), control human IgG1 (<i>n</i> = 10), or PBS (<i>n</i> = 10). Day 1 is defined at two weeks post-injection when tumors became visible. Treatments were administered 5 times (days 1, 7, 15, 25 and 34, marked by arrows) and included an initial larger dose of the proteins (20 mg/kg) followed by lower doses (10 mg/kg). Tumor progression was evaluated every three days by measuring the diameter of the tumors. The graphs show the average diameter (mm) of the tumors in each group measured in days 1–45.</p

Mechanism of NKp30-Ig mediated tumor regression.

<p>(a,b) NKp30-Ig does not induce apoptosis in tumor cells. PC3/<i>luc</i> (a) or DU145 (b) cells were incubated with increasing concentrations of NKp30-Ig, NKp46D2-Ig, control Ig or PBS in the presence of a cross-linking antibody. After 48 hours, the percentage of apoptotic cells was determined by Annexin V and PI staining. The figure shows one out of three experiments performed. (c,d) NKp30-Ig can mediate tumor opsonization by macrophages. Radioactive labeled PC3/<i>Luc</i> (c) or DU145 (d) cells were incubated with LPS-activated macrophages at the indicated E∶T ratios. Specific lysis was determined after 48 hours. Error bars represent mean±s.d of triplicates. Figure represents one out of three experiments performed. (e) Infiltration of macrophages to the tumor tissue. Human prostate tumors (DU145) grown in nude mice were fixed in 10% buffered formalin. Paraffin-embedded sections were stained in Hematoxylin and Eosin. The arrows indicate tumor associated- macrophages (X380). This figure represents one out of 5 sections tested.</p

Expression of NCRs ligands on human prostate cancer.

<p>(a,b) NKp30-Ig and NKp46D2-Ig specifically bind to human prostate cell lines. PC3/<i>Luc</i> (a) and DU145 (b) cell lines were stained with NKp30-Ig, NKp46D2-Ig or control CD99-Ig, followed by PE-conjugated mouse anti-human IgG1 antibody. Grey histograms represent the background staining by the control CD99-Ig fusion protein and the black empty histograms represent the staining by either NKp30-Ig or NKp46D2-Ig, as indicated in the top of each histogram. This figure represents one experiment out of three performed. (c) Immunohistochemical staining of primary human prostate adenocarcinoma and benign prostate hyperplasia (BPH) by NKp30-Ig and NKp46D2-Ig. Cuts from formalin-fixed and paraffin-embedded human prostate adenocarcinoma (upper panel) and BPH (lower panel) were antigen-retrieved by microwave-citrate treatment. Slides were then stained with NKp30-Ig, negative control CD99-Ig or NKp46D2-Ig, followed by biotinylated-goat-anti-human-Fc and avidin-biotin HRP complex. Substrate for HRP was AEC (red color) and slides were counter-stained with Hematoxylin. Figure shows a representative staining at X400 magnification. Arrow in NKp30-Ig staining of adenocarcinoma (top left panel) points to a representative membrane staining. Staining intensity for top left and top right panels is considered as 2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002150#s2" target="_blank">Methods</a>). (d) Expression of NKp30 and NKp46 ligands is abundant on malignant prostate tumors. Cuts from different patients suffering from benign (<i>n</i> = 8) or malignant (<i>n</i> = 9) prostate tumors were prepared and stained as above. Staining was performed in triplicates. Analysis of staining intensity (0-3) and percentage of stained tumor cells was performed by two pathologists. Positive staining was defined when staining intensity was above 1 and encompassed at least 50% of the cells, as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002150#s2" target="_blank">material and methods</a>’.</p

Summary of fusion protein treatment.

<p>(a) Visualization of tumor progression and distribution <i>in vivo</i>. The figure shows an image visualization of one representative animal of each treatment. The scale on the right of each figure describes the color map of the photon count. The integrated light emission (‘I’) is indicated in the left of each photo. (b) Summary of treatment effect. Table describes the overall effect of treatments, as shown in details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002150#pone-0002150-g003" target="_blank">figure 3</a>.</p