Absence of <i>Par1</i> accelerates MLL-AF9 driven murine leukemogenesis.

<p><b>5A</b>. Schematic overview about the performed transduction and transplantation experiments. Bone marrow isolated from <i>Par1^+/+</i> or <i>Par1^−/−</i> mice was retrovirally transduced with MLL-AF9/GFP. Equal numbers of positive cells were transplanted into lethally irradiated recipients, which were then subjected to different analyses and subsequent serial transplantations. <b>5B</b>. Survival curves of recipient mice which were transplanted with bone marrow cells of <i>Par1^+/+</i> or <i>Par1^−/−</i> mice that were retrovirally transduced with MLL-AF9 (n = 8 of each genotype). Cells of both genotypes led to a fatal leukemic disease with comparable latency. <b>5C</b>. Survival curves of secondary recipient mice which were transplanted with bone marrow cells of leukemic mice derived from the primary transplantation shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094993#pone-0094993-g005" target="_blank">Fig. 5B</a>. The secondary recipients of <i>Par1^−/−</i>;MLL-AF9 cells (n = 14) died after a significantly shorter latency than mice transplanted with <i>Par1^+/+</i>;MLL-AF9 primary blasts (n = 15; p<0.001). <b>5D</b>. The phenotypic analysis of blasts of the secondary leukemic mice did not reveal differences in CD11b expression between <i>Par1^+/+</i>;MLL-AF9 and <i>Par1^−/−</i>;MLL-AF9 cells. <b>5E</b>. <i>Par1^−/−</i>;MLL-AF9 transplanted mice (n = 8) exhibited a strong tendency towards higher percentages ofc-kit expressing cells in spleens (p = 0.055, t-test) and bone marrow (p = 0.22, t-test)compared to <i>Par1^+/+</i>;MLL-AF9 transplanted mice (n = 4).</p

PAR1 is expressed in hematopoietic cells.

<p><b>1A</b>. PAR1 was analyzed in mRNA microarray expression data from FACS sorted bone marrow cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094993#pone.0094993-Hebestreit1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094993#pone.0094993-Novershtern1" target="_blank">[23]</a>. Highest expression was found in hematopoietic stem cells (HSC) and cells of the erythroid/megakaryocyte and of the T-cell lineage. Shown here are log arbitrary units. <b>1B</b>. Left-hand side: To sort for the different murine bone marrow subpopulation, total bone marrow was stained with lineage-markers, sca1 and c-kit. Lineage-negative, sca1⁺, c-kit⁺ (LSK) cells were further divided into long-term (LT)-HSCs as Flt3⁻CD34⁻ population, short-term (ST)-HSCs as Flt3⁻CD34⁺ cells and multipotent progenitors (MPPs) as Flt3⁺CD34⁺ cells (upper panel). Common lymphoid progenitors (CLPs) were defined as lineage-negative, IL7R⁺c-kit⁺ cells. Upper and lower right panel: <i>Par1</i> mRNA expression was determined by real-time quantitative RT-PCR using cDNA from the FACS-sorted murine bone marrow subpopulations and Par1 expression was normalized to GAPDH expression. Par1 was expressed in all hematopoietic stem/progenitor subpopulations and CD3⁺ T-cells whereas monocytes/macrophages/granulocytes (CD11b⁺) or erythrocytic (Ter119⁺) or B-cells (B220⁺) expressed low or no Par1.</p

PAR1 expression in primary patient samples.

<p><b>4A</b>. Micrographs of Tissue Array analysis from NBM and AML patients stained with anti-PAR1 antibody and Fast-Red secondary antibody contrasted with hematoxylin and eosin. Overview (upper panel) and magnification of one example of CD34⁺ and AML samples that were defined PAR1-negative (lower left) and PAR1-positive (lower right). <b>4B</b>. Quantitative Tissue Array analysis of PAR1 expression using categories of staining intensity as positive or negative. Significantly more AML patient samples were negative for PAR1 expression than CD34⁺ healthy patient samples (p = 0.003, Chi-square test). <b>4C</b>. PAR1 protein was significantly less abundant in bone marrow cells from human Acute Myeloid Leukemia (AML) patients compared to CD34-positive bone marrow cells in Tissue Array samples. *p<0.05, Chi-square test.</p

Summary of non-synonymous mutations for EPHB6 (NM_004445 and NP_004436) found in tumors.

<p>Note: The table contains data from the databases of <a href="http://www.sanger.ac.uk/genetics/CGP/cosmic/" target="_blank">http://www.sanger.ac.uk/genetics/CGP/cosmic/</a>, <a href="http://strubiol.icr.ac.uk/extra/mokca" target="_blank">http://strubiol.icr.ac.uk/extra/mokca</a>, and the references were listed in the column of “Pubmed Id”. The NSCLC mutations identified in this study were marked as “not reported”. Two sequence homology-based tools were used to predict the potential impact of the identified non-synonymous substitutions on protein function: Sort Intolerant from Tolerant (SIFT; <a href="http://sift.bii.a-star.edu.sg/" target="_blank">http://sift.bii.a-star.edu.sg/</a>) and Polymorphism Phenotype (PolyPhen-2; <a href="http://genetics.bwh.harvard.edu/pph2/" target="_blank">http://genetics.bwh.harvard.edu/pph2/</a>). If the SIFT prediction tolerance index score was less than 0.05, the variation was considered possibly damaging. Predictions made by PolyPhen-2 were assigned as “probably damaging,” “possibly damaging” or “benign.” Deletion mutations cannot be tested by either SIFT or PolyPhen-2.</p

Proliferative activity and cell size of EPHB6 wildtype and mutant cells.

<p>A) Proliferative activity of empty vector control, EPHB6 wildtype and mutant cells were analyzed using a colorimetric MTT assay after 72 hours. Data are shown as means +/− standard deviation of three independent experiments. Differences were statistically not significant (ANOVA). B) Cell size of individual cells (n = 20) growing on plastic dishes was analyzed by live video microscopy and recorded. EPHB6 mutant cells showed a significantly reduced cell size in comparison to EPHB6 wild type and to control cells (p<0.05, t-test).</p

Migration analysis of EPHB6 expressing NSCLC cells.

<p>A) Protein expression of stably transfected A549 cell lines expressing wild type EPHB6 or the EPHB6 deletion mutant. Cells were co-transfected using an EGFP -pcDNA3.1⁺ vector for identification of selected clones. Multiple clones were pooled and further selected as bulk cultures. B) Transwell migration assays were performed with empty vector control cells, EPHB6 mutant and EPHB6 wildtype cells. Five different experiments in triplicates were analyzed. *: significant (p<0.05) differences by (EITHER ANOVA OR t-test) The provided p-value between the three different cell lines was statistically analyzed from all migrated cells by using the OneWay ANOVA-test. The analysis of the pair-wise t-test results in a significant p-value for the control cells vs. EPHB6-wt cells (p<0.015) and between the EPHB6-wt cells and the EPHB6-mut cells (p<0.005). C) <i>In vitro</i> wound healing scratch assay. Cells were scratched by a 10 µl pipette tip. The scratch areas were recorded over a periode of 17 hours. Shown are means of three different experiments, calculated as percentage from one initial point for all three cell lines. The ANOVA-test (p<0.002) indicated statistically significant differences between the three cell lines. D) Representative images of the scratch assays at the beginning and the end of the experiments.</p

Development of metastasis <i>in vivo</i>.

<p>A) Number of pulmonary metastases in evaluable NOD/SCID mice four weeks after transplantation, each with 3×10⁵ stably transfected A549 cells expressing EPHB6-wt (n = 9), EPHB6-del915-917 (n = 9) or empty vector control cells (n = 2). Dots represent individual mice and horizontal lines the median value of metastases. B) Images from representative whole lungs of NOD/SCID mice, transplanted with A549 cells expressing EPHB6-wt, EPHB6-del915-917, or empty vector control. Lung metastases are marked by black arrows. C) Images from lung sections of NOD/SCID mice, stained with hematoxylin. Metastases are marked by black arrows. Three representative examples are shown each for mice transplanted with A549 cells expressing EPHB6-wt or EPHB6-del915-917.</p