Fig 1: The NPY/Y5R axis contributes to filopodia formation. (A) Representative microscopic images of CHO-K1 cells transfected with EGFP alone or fused to Y2R or Y5R. (B) Representative images of CHO-K1/Y2R-EGFP and CHO-K1/Y5R-EGFP cells stained with Texas Red®-X phalloidin. (C) Frequency of cells with multiple filopodia (10 or more per cell) in CHO-K1/EGFP, CHO-K1/Y2R-EGFP, and CHO-K1/Y5R-EGFP cell lines. (D) A representative image of CHO-K1/Y5R-EGFP cells with multiple filopodia. (E) Effect of NPY on the frequency of CHO-K1/Y5R-EGFP cells with multiple filopodia. (F) A representative image of SK-N-AS NB cells with Y5R-postive and Y5R-negative filopodia stained with anti-Y5R antibody (green) and phalloidin (red). (G) Frequency of Y5R-positive filopodia in SK-N-AS cells with or without NPY (10−9-10−7 M). Panels (C,E,G): *p < 0.05, ***p < 0.001; ****p < 0.0001 by Chi-square test; results shown as a percent of cells (C,E) or filopodia (G); n = 25 per condition. CHO-K1/Y5R-EGFP and SK-N-AS shown and analyzed in (C–G) were cultured in wound healing assay chambers, and only cells at the migratory edges of wounds were analyzed.
Fig 2: NPY/Y5R axis is involved in cell invasiveness. (A) Representative microscopic image of CHO-K1/Y5R cells invading Matrigel. White arrows indicate invadopodia. (B) NPY-induced SK-N-BE(2) cell invasiveness measured in a Matrigel-coated Transwell plate. The cells were subjected to NPY (10-11-10-7 M) for 22 h in the lower chamber of the Transwell plate (directional invasiveness) or both upper and lower chambers (spontaneous invasiveness). *p < 0.05 by one-way ANOVA followed by Dunnett's test; mean ± SEM, n = 4.
Fig 3: In CHO-K1 cells, overexpression of Y5R promotes cellular migration, while Y2R inhibits cell motility. Migration of CHO-K1 cells transfected with Y2R or Y5R fused to EGFP or with EGFP alone, measured by a wound healing assay with IncuCyte ZOOM live-cell imaging system analysis. Representative images captured by the IncuCyte software followed by quantitative analyses are shown. (A) Cell migration in 10% FBS. (B) Cell migration in 1% FBS. The migration of CHO-K1/Y5R-EGFP cells measured in the presence or absence of Y5R antagonist (CGP71683, 10-6 M). (C) Migration of CHO-K1/EGFP and CHO-K1/Y5R-EGFP cells in response to NPY (10-10-10-7 M) in 0.1% FBS. (A–C) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 as shown or ####p < 0.0001 vs. CHO-K1/EGFP under the same conditions by one-way ANOVA followed by Tukey's test; mean ± SEM from three independent experiments, n = 3–32 each.
Fig 4: In situ proximity ligation assay (PLA) reveals interactions between Y5R and RhoA-GTP. PLA performed in SK-N-AS cells with anti-Y5R and anti-RhoA-GTP antibodies. (A) Representative confocal microscopy images of PLA in SK-N-AS cells treated with NPY (10-8 M) for 5, 10, and 20 min. Red dots represent interactions between Y5R and RhoA-GTP. The graph depicts quantification of fluorescence intensities for the total PLA signal or the signal located inside of the cell colonies and near their outer membranes in SK-N-AS cells treated with NPY. (B) PLA (red) and phalloidin staining (green) in SK-N-AS cells treated with NPY (10-8 M) for 20 min, with or without Y5R antagonist (10-6 M) pre-treatment for 30 min. White arrows indicate leading edges of the neuroblastoma (NB) cells. The graph represents a quantification of fluorescence intensity of the PLA signal—total or located inside of cell colonies and near their outer membranes in SK-N-AS cells treated with NPY and Y5R antagonist as above. (A,B) *p < 0.05, **p < 0.01, ****p < 0.0001 vs. non-treated control or ****p < 0.0001 as indicated, by one-way ANOVA followed by Tukey's test; mean ± SEM, n = 11–21 per condition. (C) Co-localization of the PLA signal (red) with F-actin (green) on the outer membranes of cells within the colony with migratory phenotype. (D) PLA signal in the area rich in F-actin near the outer plasma membrane. (E) Localization of the PLA signal along F-actin fibers.
Fig 5: The NPY/Y5R pathway activates RhoA. (A) Immunocytochemistry with anti-Y5R (green) and anti-RhoA-GTP (red) antibodies in CHO-K1/Y5R-EGFP cells. (B) RhoA pull-down assay in CHO-K1/Y5R-EGFP cells treated with NPY (10-7 M). (C) RhoA pull-down assay in SK-N-AS NB cells treated with NPY (10-10-10-7 M). (D) Quantitative analysis of RhoA activity measured by a pull-down assay in SK-N-AS cells treated with NPY at a concentration of 10-8 M. (C,D) *p < 0.05 by t-test; mean ± SEM from three independent experiments.
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