a The TGF-β2 secretion level in cell culture medium was confirmed by western blotting. Total proteins staining with Coomassie Brilliant Blue were used as the loading control. b Western blotting was performed to determine the expression of TGF-β2, PI3K, phosphorylated-PI3K (p-PI3K), AKT, phosphorylated-AKT (p-AKT), STAT3, pY705STAT3 (p-STAT3), NANOG, c-Myc, c-Jun, and BCL-XL in cell lysates from 8024-Vec/RALYL, MIHA-Vec/RALYL, Huh7-Control/shRALYLs, and H2M-Control/shRALYLs. c PLC-8024 and MIHA cells were treated with conditioned medium from 8024-Vec/RALYL or MIHA-Vec/RALYL, respectively (Ctrl CM: conditioned medium from Vec transfected cells; R CM: conditioned medium from RALYL transfected cells). Cell lysates were analyzed using western blotting to compare the expression levels of AKT, p-AKT, STAT3, p-STAT3, c-Myc, and c-Jun. d PLC-8024 and Huh7-shRALYL4 were treated with TGF-β2 at 2 or 10 ng/mL; 8024-RALYL cells were treated with TGF-β2-neutralizing antibody at 1 μg/mL. The expression levels of AKT, p-AKT, STAT3, p-STAT3, c-Myc, and c-Jun were determined by western blotting. e The XTT assay was used to assess the effect of the STAT3 inhibitor (NSC74859) on the proliferation of 8024-Vec/RALYL (NSC74859: 40 μM) and Huh7-Control/shRALYL cells (NSC74859: 20 μM). The values represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). f Representative images of tumors induced by indicated cells in nude mice treated with NSC74859 once 3 days for 3 weeks. The average tumor volume was expressed as mean ± SD of five mice.