a An in vitro hepatocyte differentiation model, which induced human embryonic stem (ES) cells into definitive endoderm (DE), liver progenitor cells (LP) and premature hepatocytes (PH) step by step, was established. Two HCC samples and two normal liver tissues (NL) were also used to perform the transcriptome sequencing. b Heatmap of expression profiles for the specific markers for ES (Oct4, Nanog, and Sox2), DE (Sox17, FOXA2, Nodal, and Hhex), liver progenitor cells (AFP, Gata3, HNF4α, and FOXA1), and hepatocyte (Albumin, FOXA3, Hlx, and Cyp3a4) shows the reliability of the hepatic differentiation model. The red represents a higher expression level, and the green represents a lower expression level. c RT-PCR analysis showed higher RALYL expression in CD133+ cells than in CD133− cells, which was sorted by FACS from Huh7 or Hep3B cells. d The proportions of CD133+ cells in 8024-Vec/RALYL and MIHA-Vec/RALYL were assessed by flow cytometry and illustrated in bar chart. The values indicate the mean ± standard deviation (SD) of three independent experiments (**P < 0.01, Student t-test). e Representative images of double immunofluorescence staining of RALYL (green) and CD133 (red) in 8024-Vec/RALYL and MIHA-Vec/RALYL. DAPI (blue) was used for nuclei counterstaining. Scale bar = 10 μm. f Kaplan–Meier overall survival curve and disease-free survival curve of two HCC groups in our in-house cohort or The Cancer Genome Atlas (TCGA) cohort: RALYL(+) (red), patients with RALYL expression; RALYL(–) (blue), patients without RALYL detection.