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

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 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