(A) Real-time cell proliferation analysis of HCC cells (PLC) in serum-containing (i.e., NPY-containing) culture medium applying an NPY-neutralizing antibody (Anti-NPY Ab) (n = 2; 2 replicates per independent experiment). (B) Clonogenicity assay (representative images and quantification of colony sizes) of NPY-treated (500 nM) and control-treated HCC cells (PLC) applying serum-free medium (n = 4). (C and D) Western blot analysis of phospho-ERK (p-ERK) and ERK levels (representative images and densitometric quantification) of HCC cells (PLC) treated with recombinant NPY with or without combined treatment applying the specific Y5R inhibitor CGP71683 (Y5R-Inh) (n = 3) (C) (data are shown as box-and-whisker plots [min to max]), or treated with the specific Y5R agonist (BWX46) (the representative image shows duplicates for Y5R agonist treatment) (n = 3) (D). (E) Real-time cell proliferation analysis of cocultured HCC cells and PHHs applying the Y5R inhibitor or a neutralizing NPY antibody (Anti-NPY Ab) as compared with control treatment (n = 8). (F) Western blot analysis (and representative image) of p-ERK and ERK levels in HCC cells incubated with cell culture supernatant of PHHs (containing NPY as quantified by ELISA) with or without combined treatment applying Y5R-Inh or anti-NPY Ab (n = 3) (data are shown as box-and-whisker plots [min to max]). Data are presented as mean ± SEM. Statistical significance was determined by ordinary 1-way ANOVA together with Dunnett’s multiple-comparisons test (C, D, and F), or by 2-tailed, unpaired t test (A, B, and E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.