a RT-PCR and western blotting showed ectopic expression of RALYL in cells transfected with RALYL or control plasmid, and RALYL silencing in cells treated with scrambled shRNA (Control: Ctrl) or shRNA against RALYL (shRALYL-1: sh1; shRALYL-4: sh4). 18S and β-actin were used as loading controls. b XTT assay was used to determine the cell proliferation rates. The values indicate the mean ± SD of three independent experiments. P value was shown as *(P < 0.05) or **(P < 0.01) at some time points and highlighted in the color same with the line of corresponding experiment group (* in purple: 8024-RALYL vs. 8024-Vec, MIHA-RALYL vs. MIHA-Vec, Huh7-shRALYL1 vs. Huh7-Control, Hep3B-shRALYL1 vs. Hep3B-Control; * in red: Huh7-shRALYL4 vs Huh7-Control, Hep3B-shRALYL4 vs. Hep3B-Control, independent Student’s t-test). Representative images of foci formation (c) and colony formation in soft agar (d) induced by 8024-Vec/RALYL, MIHA-Vec/RALYL, Huh7-Control/shRALYL and Hep3B-Control/shRALYL. The numbers of foci and colonies are illustrated in bar chart. The values indicate the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01, two-sided Student’s t-test). e Representative images of mice with tumors induced by the indicated cells. Weights of tumors are expressed as mean ± SD of five mice (independent Student’s t-test).