NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.

<p><b>(A)</b> Representative images. <b>(B, D)</b> Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. <b>(C, E)</b> Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ² tests followed by Fisher’s exact tests. <b>(F)</b> Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ² tests followed by Fisher’s exact tests. <b>(G)</b> Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.</p

Association of NF-κB expression with clinical and pathologic parameters in 77 patients with NSCLC.

<p><b>(A)</b> NF-κB expression levels subdivided by clinical and pathological parameters. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, *, and **: P > 0.05, P < 0.05, and P < 0.0501 for indicated comparisons by Wilcoxon signed rank tests or Kruskal-Wallis tests followed by Dunn’s post-tests, for two or multiple comparison groups, respectively. <b>(B)</b> Results of binary logistic regression analyses using NF-κB subunit expression scores as the input (independent variables) and dichotomized clinical and pathologic parameters as the output (dependent variables). RR, risk ratios; CI, confidence intervals; P, probability values.</p

Immunohistochemical detection of NF-κB in mouse models of NSCLC.

<p>NF-κB subunit expression was assessed by immunohistochemistry in urethane-induced mouse lung adenomas <b>(A and C)</b> and mutant <i>KRAS</i>-induced lung adenocarcinomas <b>(B and D)</b>. <b>(A, B)</b> Representative images. <b>(C, D)</b> Overall scoring of NF-κB subunit expression levels from four mice per group. Data presented as mean ± SD. ** and ***: P < 0.01, and P < 0.001 for the indicated color-coded subunit compared with normal bronchial and alveolar epithelium by two-way ANOVA followed by Bonferroni post-tests. Non-significant comparisons are not indicated.</p

Immunohistochemical detection of NF-κB subunits in NSCLC, juxta-tumoral normal lung structures and preneoplastic lesions.

<p><b>(A)</b> Representative images. Images in red frames representatively display differential NF-κB subunit expression in tumor and intratumoral stroma areas. <b>(B)</b> Overall scoring of NF-κB subunit expression levels. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, * and ***: P > 0.05, P < 0.05, and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. <b>(C)</b> Co-expression matrixes of categorical NF-κB subunit expression levels. For this, NF-κB scores from (B) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 by χ² tests followed by Fisher’s exact tests.</p

Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.

<p><b>(A)</b> Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. <b>(B)</b> NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. <b>(C)</b> Representative images of PCNA-stained NSCLC subtype samples. <b>(D)</b> Nuclear co-localization of PCNA immunoreactivity with <i>Rel</i>B (arrows), but not with <i>Rel</i>A, was identified using dual immunostaining of samples of 10 patients (representative images shown).</p

Schematic illustration of the main findings of the present study.

<p>NF-κB subunit expression levels in tumor and stroma cells of 77 patients with NSCLC are indicated by relative font size. Arrows indicate possible associations of <i>Rel</i> protein expression levels in NSCLC tumor cells with tumor-associated inflammation and cellular proliferation.</p

Insights into Soluble Guanylyl Cyclase Activation Derived from Improved Heme-Mimetics

Recently, the structure of BAY 58-2667 bound to the Nostoc sp. H-NOX domain was published. On the basis of this structural information, we designed BAY 58-2667 derivatives and tested their effects on soluble guanylyl cyclase (sGC) activity. Derivative <b>20</b> activated sGC 4.8-fold more than BAY 58-2667. Co-crystallization of <b>20</b> with the <i>Ns</i> H-NOX domain revealed that the increased conformational distortion at the C-terminal region of αF helix containing 110–114 residues contributes to the higher activation triggered by <b>20</b>

CCL2 and/or CCL12 neutralization inhibits MPE-precipitating vascular hyperpermeability.

<p>(A,B) Pleural fluid Evans’ blue levels from mice with LLC- and MC38-induced MPE treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071207#pone-0071207-g001" target="_blank">Figure 1A</a>. Mice received intravenous Evans’ blue before sacrifice, followed by quantification of the albumin tracer in MPE. *, ** and ***: P<0.05, P<0.01, and P<0.001 compared with saline and/or IgG2a. (C) Summary of data (<i>n</i> = 5) and photographs of representative skin test sites from C57BL/6 mice that received intradermal injections of IgG2a or cell-free MPE fluid admixed with IgG2a or anti-CCL2/12 antibodies, followed immediately by intravenous delivery of Evans’ blue. Mice were euthanized after one hour, followed by skin inversion and imaging. Shown is test spot area relative to the control test spot on the same mouse. ##: P<0.01 compared with IgG2a; * and **: P<0.05 and P<0.01 compared with MPE. <i>Columns</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size.</p

CCL2 and/or CCL12 blockade impact on tumor growth.

<p>(A) Pleural tumors from mice with LLC-induced MPE treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071207#pone-0071207-g001" target="_blank">Figure 1A</a> were stained with Hoechst 33258, anti-Caspase-3, and PCNA antibodies. Shown are summary of data for PCNA staining (left) and representative images from pleural tumors of an IgG2a and a combination-treated mouse (right). Scale bar = 100 µm, Å = 400. Arrows indicate rare caspase-3 positive cells. (B,C) Volume of flank tumors induced by subcutaneous injection of LLC and MC38 cells after treatment with IgG2a control or anti-CCL2/CCL12 combination therapy. Arrows indicate the day of antibody therapy start. (D) Volume of flank tumors induced by subcutaneous injection of LLC cells stably expressing random or anti-CCL2-specific shRNAs (sh166 and sh436). (E) Tumor volumes from flank tumor experiments at four weeks. (F) Photograph of mouse thoracic-abdominal border (dashed line) four hours after intraperitoneal Evans’ blue delivery. <i>Columns and squares</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size; ns and ***, P>0.05 and P<0.001 compared with saline and/or IgG2a or random shRNA controls.</p

CCL2 and/or CCL12 neutralization impacts new vessel assembly in pleural tumors.

<p>(A) Microvessel density of pleural tumors from mice with LLC- and MC38-induced MPE treated with IgG2a or anti-CCL2/12 combination as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071207#pone-0071207-g001" target="_blank">Figure 1A</a>. Shown is summary of data and representative images of factor-VIII-associated protein (F8A) immunoreactivity. Scale bar = 100 µm; Å = 400. Arrows indicate new vessels. (B) Representative chorioallantoic membranes and summary of data obtained from six membranes/group that were incubated with IgG2a or cell-free MPE fluid admixed with IgG2a or anti-CCL2/12 antibodies. Scale bar = 5 mm. <i>Columns</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size; ns: P>0.05; #: P<0.05 compared with IgG2a; *, **, and ***, P<0.05, P<0.01, and P<0.001 compared with MPE.</p