Ultrastructural immunocytochemistry detection of ANXA1 and FPR2/ALX co-localization in laryngeal cells by electron microscopy.

<p>Immunolabeling with 10-nm (FPR2/ALX) and 15-nm (ANXA1) colloidal gold particles. (<b>A–C</b>) Epithelial control, peritumoral and tumor cells from laryngeal tissues. (<b>D–E</b>) Hep-2 cells showing immunoreactivity to ANXA1 (arrows), FPR2 (arrowheads), and co-localizations (curve arrows) in the plasma membrane, cytoplasm and nucleus (N). Desmosomes (open curve arrows) are detected between these cells. (<b>F</b>) The absence of immunoreactivity to ANXA1 and FPR2/ALX in cells incubated with nonimmune serum. Density of gold particles conjugated with ANXA1 and FPR2/ALX in epithelial cells (<b>G–I</b>) and Hep-2 cells (<b>J–L</b>). Hep-2 cells were seeded in MEM-Earle medium at a density of 2×10⁶ cells in 75-cm² culture flasks, and then were incubated with serum-free medium 24 hours prior to the addition of ANXA1_2–26 (1 µM) and ANXA1_2–26 (1 µM)+Boc2 (10 µM). Data are expressed as the mean ± SEM of colloidal gold particles per µm in the plasma membrane and per µm² in the cytoplasm and nucleus of cells (n = 10 cells per group). One-way ANOVA followed by Bonferroni's test revealed a significant difference among the groups. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 <i>vs.</i> control, # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001 <i>vs.</i> ANXA1_2–26. Stained with uranyl acetate and lead citrate. Scale bars: 1 µm.</p

Effect of the peptide ANXA1_2–26 and antagonist Boc2 on the proliferation of Hep-2 cells.

<p>Treatment with ANXA1_2–26 reduced the cellular growth. The antagonist Boc2 partially inhibited the anti-proliferative effect of ANXA1_2–26. The cells treated with Boc2 alone showed a level of proliferation similar to that of the control. The Hep-2 cells were seeded in MEM-Earle medium at a density of 2×10⁶ cells in 75-cm² culture flask, and then were incubated with serum-free medium 24 hours prior to the addition of ANXA1_2–26 (1 µM), ANXA1_2–26 (1 µM)+Boc (10 µM) or Boc (10 µM) alone. All of the experiments were performed in triplicate to confirm the results. Data are expressed as the mean ± SEM of the cell number ×10⁶. ** <i>P</i><0.01 and *** <i>P</i><0.001 <i>vs.</i> control, ## <i>P</i><0.01 and ### <i>P</i><0.001 <i>vs.</i> ANXA1_2–26+Boc.</p

Effect of the peptide ANXA1_2–26 on proinflammatory cytokine expression.

<p>Low expression of IL-6 (<b>A</b>), IL-8 (<b>B</b>) and MCP-1 (<b>C</b>) after treatment with ANXA1_2–26 and dexamethasone. Hep-2 cells were seeded in MEM-Earle medium at a density of 2×10⁶ cells in 75-cm² culture flasks, and then were incubated with serum-free medium 24 hours prior to the addition of ANXA1_2–26 (1 µM), ANXA1_2–26 (1 µM)+Boc2 (10 µM), Dexa (0.01 µM), Dexa (0.01 µM)+Boc2 (10 µM) or Boc2 (10 µM) alone. All of the experiments were performed in triplicate to confirm the results. Data are expressed as the mean ± SEM of the analyte concentration (pg/mL), determined using <i>MAGPIX xPONENT software</i>. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 <i>vs.</i> control, ## <i>P</i><0.01 and ### <i>P</i><0.001 <i>vs.</i> ANXA1_2–26, §§ <i>P</i><0.01 and §§§ <i>P</i><0.001 <i>vs.</i> ANXA1_2–26+Boc2.</p

Immunoreactivity to ANXA1 and FPR2/ALX in mast cells and neutrophils of laryngeal sections.

<p>Immunolabeling with 10-nm (FPR2/ALX) and 15-nm (ANXA1) colloidal gold particles. Mast cells (<b>A–C</b>) and neutrophils (<b>D–F</b>) showing subcellular localization of ANXA1 (arrows) and FPR2/ALX (arrowheads), and co-localization (curve arrows) in the plasma membrane, cytoplasm and nucleus (N) of control, peritumoral and tumor tissues. Density of gold particles conjugated with ANXA1 and FPR2/ALX in mast cells (<b>G–I</b>) and neutrophils (<b>J–L</b>). Data are expressed as the mean ± SEM of colloidal gold particles per µm in the plasma membrane and per µm² in the cytoplasm and nucleus of cells (n = 10 cells per group). One-way ANOVA followed by Bonferroni's test revealed a significant difference among the groups. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 <i>vs.</i> control, ## <i>P</i><0.01, ### <i>P</i><0.001 <i>vs.</i> peritumoral. Stained with uranyl acetate and lead citrate. Scale bars: 1 µm.</p

Multivariate analysis of the relationship between clinical and pathological tumor features with gene polymorphism and FGFR4 expression.

a, b<p>Values adjusted by multivariate logistic regression.</p><p>For Gly388Arg and FGFR4 expression correlation with lymphatic embolization and lymph node status, tumor size and differentiation status were considered in the multivariate analysis. For disease relapse and disease specific death, tumor size, lymph node status and radiotherapy treatment were considered.</p

Immunohistochemical analysis of tumors.

<p>(a) strong FGFR4 expression; (b) weak FGFR4 expression. Magnification was 400×.</p

Epidemiological, clinical and pathological tumor features and their association with Gly388Arg polymorphism and FGFR4 expression.

a<p>Not available (not considered in the statistical calculations).</p

Survival plots.

<p>a. and b.: Disease-free survival and disease specific survival according to FGFR4 Gly388Arg polymorphism; c. and d.: Disease-free survival and disease specific survival according to FGFR4 expression; e. and f.: Disease-free survival and disease specific survival according to FGFR4 profile.</p

Survival plots.

<p><b>a. and b.:</b> Disease-free survival and disease-specific survival according to FAS/FASL profile.</p

Epidemiological, clinical and pathological tumor features and their association with FAS and FASL expression.

¥<p>TNM classification 3^rd edition.</p>*<p>Not available (not considered in the statistical calculations).</p