Fig 2: 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 3: Surface Y5R is distributed in the areas of cytoskeleton remodeling in migratory CHO-K1/Y5R-EGFP cells. Immunocytochemistry with anti-Y5R antibody (green) in non-permeabilized CHO-K1/Y5R-EGFP cells counterstained with phalloidin (red) to detect F-actin. In all panels, white arrows indicate Y5R expression in the following regions: (A) Polar distribution of Y5R. (B) Y5R at leading edges of the migratory cells. (C) Y5R at the trailing edge of a migratory cell. (D) Y5R in filopodia and the areas of their formation. (E) Y5R in defined areas of the long CHO-K1/Y5R-EGFP cell protrusions. (F) Representative images of Y5R at the leading edges of the migrating CHO-K1/Y5R-EGFP cells via the IncuCyte ZOOM live-cell imaging system (0–23 h).

Fig 4: 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 5: The NPY/Y5R axis promotes migration in NB cells. (A) Western blot analysis of Y5R expression in SK-N-AS and SK-N-BE(2) NB cell lines. *p < 0.05 by paired t-test, mean ± SEM, n = 4. (B) Spontaneous migration of SK-N-BE(2) cells treated for 22 h with NPY (10-7 M), in the presence or absence of Y2R antagonist (BIIE0246) and Y5R antagonist (CGP71683), each at a concentration of 10-6 M. (C) SK-N-AS cell migration in response to NPY (10-7 M), with or without Y2R and Y5R antagonists (10-6 M). (B,C) Spontaneous migration measured by a Transwell assay with NPY in both upper and lower chambers. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey's test; mean ± SEM from two independent experiments, n = 4 each.