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

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

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

Prognostic analysis for suspicious radiologic findings (<i>i</i>.<i>e</i>. SPN, LAP and PE) in patients under TAVI evaluation with complete follow-up data (n = 237 patients).

<p>Prognostic analysis for suspicious radiologic findings (<i>i</i>.<i>e</i>. SPN, LAP and PE) in patients under TAVI evaluation with complete follow-up data (n = 237 patients).</p

Baseline characteristics of patients under TAVI evaluation with complete follow-up data (n = 237 patients).

<p>Baseline characteristics of patients under TAVI evaluation with complete follow-up data (n = 237 patients).</p

Prognostic impact of left ventricular ejection fraction (LVEF) in the full study collective (p = 0.004).

<p>Overall survival of those patients with a LVEF ≥ 45% was increased compared to those patients with a LVEF < 45%.</p

The incidence of incidentally discovered SPN (cases on chest computer tomographies) and the number of diagnosed lung cancer cases.

<p>The incidence of incidentally discovered SPN (cases on chest computer tomographies) and the number of diagnosed lung cancer cases.</p

Prognostic impact of solitary pulmonary nodules (SPN), lymphadenopathy (LAP) and pleural effusions (PE) in patients under evaluation for TAVI (n = 237 patients).

<p>Kaplan Meier charts are given for SPN ≥ 5mm (<b>A</b>), for SPN > 8mm (<b>B</b>), for LAP (<b>C</b>) and for PE (<b>D</b>).</p

Correlations of clinical and respiratory parameters with suspicious radiologic findings (<i>i</i>.<i>e</i>. SPN, LAP and PE) for patients under TAVI evaluation with complete follow-up data (n = 237 patients).

<p>Correlations of clinical and respiratory parameters with suspicious radiologic findings (<i>i</i>.<i>e</i>. SPN, LAP and PE) for patients under TAVI evaluation with complete follow-up data (n = 237 patients).</p