Fig 2: LAGE3 promotes the progression of HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways. A The mRNA levels of PI3CA, BRAF, AKT, MAP2K1, ERK, and KRAS in the siLAGE3 group were detected by qRT-PCR expressing the data as the mean ± s. d., n. s: no significant difference, *** p < 0.001. B The protein levels of PI3K, RAF, MUC1, p-Akt, p-MEK1, p-Erk, and Ras were significantly down-regulated in the siLAGE3 group while data were expressed as the mean ± s. d., ** p < 0.01 and *** p < 0.001. The overexpression efficiency of LAGE3 by LAGE3 gene lentivirus particles infection and the empty plasmid lentivirus particles in Huh7 cells was verified by Western blot (C) and qRT-PCR (D), where the data were expressed as the mean ± s. d., *** p < 0.001. The proliferation of Huh-7 cells was significantly enhanced in the overexpression of BRAF in (E) MTT assay and Brdu assay (F), while data were expressed as the mean ± s. d., * p < 0.05. G The Huh-7 cell cycle was significantly arrested in the S and G2/M phase in the BRAF overexpression, where the data were expressed as the mean ± s. d., * p < 0.05. H The proportion of apoptotic cells was significantly reduced in the BRAF overexpression group as assessed by flow cytometry and expressing the data as the mean ± s. d., * p < 0.05

Fig 3: Overexpression of LAGE3 in HCC tissues and cell lines. A Usage of GEPIA2 tool to perform OS and DFS analyses of LIHC tumors in TCGA by LAGE3 gene expression for obtaining Kaplan-Meier curves. B The LAGE3 expression analyses in LIHC tumors as included in TCGA were performed by the GEPIA2 tool and the box plot data were supplied. * p < 0.05. C High expression of LAGE3 mRNA level in HCC cell lines while the data were expressed as the mean ± s. d., *** p < 0.001. D IHC staining images of LAGE3 expressions in HCC tumor tissue chips. OS: Overall survival; DFS: Disease-free survival; LIHC: liver hepatocellular carcinoma; IHC: Immunohistochemistry

Fig 4: LAGE3 knockdown suppressed HCC cell migration and invasion. A and B Migration of Huh-7 and SMCC-7721 cells was significantly inhibited by LAGE3 knockdown in scratch healing assay expressing the data as the mean ± s. d., * p < 0.05 and *** p < 0.001. C and D Migration of Huh-7 and SMCC-7721 cells were significantly inhibited by LAGE3 knockdown in Transwell assay expressing the data as the mean ± s. d., * p < 0.05 and ** p < 0.01. E and F while the invasion of Huh-7 and SMCC-7721 cells were significantly inhibited by LAGE3 knockdown in Transwell assay expressing the data as the mean ± s. d., ** p < 0.01 and *** p < 0.001

Fig 5: LAGE3 knockdown promoted HCC cells apoptosis. The proportion of apoptotic cells was significantly increased in the LAGE3 knockdown group compared with control cells in Huh7 (A) and SMCC-7721 (B) cell lines as assessed by flow cytometry thus expressing the data as the mean ± s. d., *** p < 0.001