(A) ELISA analysis of NPY protein levels in cell culture medium with FCS (n = 3), serum-free medium (Control; n = 3), and cell culture supernatants of primary human hepatocytes (PHH; n = 6), human fibroblasts (HF; n = 2), and hepatic stellate cells (HSC; n = 3). (B) Representative images of (co)immunofluorescence staining (NPY and α-SMA; 20-fold original magnification) of peritumorous liver tissues of C3H mice (left, n = 3) and cultures of PHHs or HSCs (n = 3; right). (C) NPY mRNA of PHHs treated or not treated with conditioned culture medium derived from HSCs (HSC-CM; n = 6). (D) NPY protein levels in cell culture supernatants treated or not treated with HSC-CM (n = 3). (E) NPY mRNA in PHHs treated for 48 hours with different doses of TGF-β (n = 3). (F) NPY mRNA in hepatocyte-derived liver organoids treated for 48 hours (n = 10) or 96 hours (n = 4) with TGF-β (left). Representative images of qRT-PCR gel electrophoresis (n = 3) and confocal immunofluorescence (60-fold original magnification; n = 2) (right). (G) Representative IHC TGF-β staining of (human) peritumorous liver (40-fold original magnification). (H–J) IHC analysis of TGF-β and NPY in peritumorous liver and corresponding HCC tissues. (H) Representative images (40-fold original magnification). (I) Comparison of TGF-β expression in HCC and corresponding peritumorous liver tissues (n = 219). (J) Comparison of NPY expression in peritumorous liver tissues with low (n = 38) and high (n = 63) TGF-β expression. Data are presented as mean ± SEM. Statistical significance was determined by ordinary 1-way ANOVA together with Dunnett’s multiple-comparisons test (D–F) or 2-tailed, unpaired t test (C), or by 2-sided Fisher’s exact test and Spearman’s correlation analysis (I and J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.