(A) Chemical formula of the specific, high-affinity Y5R inhibitor CGP71683 (Y5R-Inh). (B–D) Y5R-Inh–treated as compared with control-treated (i.e., solvent [DMSO]) HCC cells with application of different functional in vitro assays. (B) FACS analysis (representative images and quantification) after propidium iodide (PI) staining; quantification of sub-G₁/G₀, G₁, S, and G₂ cell cycle fractions (n = 3) (data are shown as box-and-whisker plots [min to max]). (C) Real-time proliferation analysis applying different doses of Y5R-Inh (n = 3) (data are shown as box-and-whisker plots [min to max]). (D) Quantification of colony numbers and sizes and representative images with application of clonogenicity assays (n = 4). (E and F) Murine HCC models of orthotopic syngeneic HCC cell implantation (Hepa129 cells) in C3H mice. (E) After HCC cell implantation, mice were randomized into 2 groups and were treated with Y5R-Inh (15 mg/kg body weight, daily i.p. injection for 7 days) or received the solvent only. (F) For RNAi-mediated Y5R knockout, transfection of HCC cells was performed for 48 hours before injection into livers, and tumor onset was assessed after 7 days. Data are presented as mean ± SEM. Statistical significance was determined by ordinary 1-way ANOVA together with Dunnett’s multiple-comparisons test (C and D) or 2-tailed, unpaired t test (B and E), or by 2-sided Fisher’s exact test together with Spearman’s correlation analysis (F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.